JP2020523037A - Allotolerance without the need for systemic immunosuppression - Google Patents

Abstract

Provided are cells that have been genetically modified to include at least one mechanism for providing local immunosuppression at the site of transplantation when transplanted into a cognate host, and methods of making and using the cells. A cell is a set of transgenes, each transgene is a gene product that is cytoplasmic, membrane-bound or locally acting, the function of which is to relax activation and function of antigen presenting cells. , Alleviating graft-attacking leukocyte activity or cytolytic function, alleviating macrophage cytolytic function and phagocytosis of allograft cells, inducing apoptosis in graft-attacking leukocytes, mitigating local inflammatory proteins , And a set of transgenes encoding gene products that are one or more of protecting against leukocyte-mediated apoptosis. The transgene comprises two or more of the following genes: PD-L1, HLAG or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8 and Serpin B9 or Spi6. [Selection diagram] Figures 1A-D

Description

The present disclosure relates generally to the field of transplantation. The disclosure further relates to methods for producing local immunosuppression in the environment of transplanted cells.

The advent of human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells has a paradigm shift effect on regenerative and translational medicine. These cells can self-renew indefinitely in a pluripotent state while retaining the ability to differentiate into any cell type in the human body. Such characteristics have allowed researchers to better understand the etiology of human development and developmental disorders. Researchers also have powerful new tools for diseases that were difficult or impossible to treat with conventional medicine, including spinal cord injury, diabetes, blindness, multiple sclerosis and cancer, to name a few. Given to modern medicine. The efficacy and extent of disease applicable to cell therapy only increases with an increasing understanding of how to control stem cell differentiation and the biology of differentiated cell products.

These applications present significant and significant challenges. In addition to cell safety, one of the most important concerns is the immune rejection of cells from different genetic backgrounds. The immune system recognizes "non-self" cells that express specific protein fragments (especially those from the major histocompatibility complex (MHC in mouse, HLA in human)) that differ between donor and recipient and Immune rejection remains an important barrier because of the evolution of a complex set of mechanisms for elimination (Yang et al., Nat Rev Genet. 18:309-26 (2017)). It is almost certain that this response is a by-product of evolutionary pressure to protect against opportunistic infections and malignancies (often defined by the presence of "foreign" proteins and epitopes). Depending on the context, rejection of transplanted cells or tissues can occur over minute/hour (hyperacute), day/month (acute) and month/year (chronic) time scales (LaRosa et al., J. Immunol. 178:7503-9 (2007)). This rejection is derived from both innate (Murphy et al, Immunol Rev. 241:39-48 (2011)) and adaptive immunity (Issa et al., Expert Rev Clin Immunol. 6:155-69 (2010)). Due to the complex and coordinated effects of the cell types involved.

One of the most important pathways for rejection is priming of the adaptive immune system and activation of CD8+ cytotoxic T cells. This occurs after antigen-presenting cells process the donor-specific peptide and then activate recipient T cells that are specific for the same peptide in secondary lymphoid organs (Lechler et al., J Exp Med. 155: 31-41 (1982); Guermonprez et al., Annu Rev Immunol. 20:621-67 (2002); Stockwin et al., Immunol Cell Biol. 78:91-102 (2000)). These T cells then migrate to the transplanted cells or tissues, killing them with the release of cytolytic factors such as perforin and granzymes. NK cells may also induce apoptosis in donor cells based on exogenous MHC expression or no MHC expression (Kitchens et al., Transplantation. 81: 811-7 (2006); Benichou et al., Curr Opin Organ Transplant. 16). : 47-53 (2011)), other cell types such as macrophages may support rejection with release of proinflammatory cytokines at the site of transplantation (Mannon, Curr Opin Organ Transplant. 17:20-5( 2012)). Many other cell types and subtypes also have a role in allograft rejection. They are highly conserved across vertebrate species with allograft rejection because they are the same immune pathways used to eliminate common viral and bacterial pathogens.

Current solutions to prevent allograft rejection include two options: finding donors with matched histocompatibility haplotypes (mostly from genetically related families). , And more generally broadly directed use of immunosuppressive drugs (Wiseman, Clin J Am Soc Nephrol. 11:332-43 (2016); Malaise et al., Transplant Proc. 37:2840-2 (2005)). Examples of common drugs include calcineurin inhibitors (Flechner et al., Clin Transplant. 22:1-15 (2008); Casey et al., Curr Opin Nephrol Hypertens. 20:610-5 (2011)), antiproliferative agents. Agent (Hardinger et al., World J Transplant. 3:68-77 (2013)), mTOR inhibitor (Macdonald, Expert Rev Clin Immunol. 3:423-36 (2007); Neuhaus et al., Liver Trans. : 473-84 (2001)) and steroids (Steiner et al., Semin Immunopathol. 33:157-67 (2011)) family, all of which suppress proliferation or function of T cells. Do (especially the first three). These drugs need to be taken daily for life, and forgetting one dose may increase the risk of rejection. However, they do not always work, and if they do, the rate of chronic rejection still rises continuously over time (Demetris et al., Ann Transplant. 2:27-44 (1997). ); Libby et al., Immunity. 14:387-97 (2001)). Most importantly, they are systemically active, ultimately rendering patients immunodeficient with an increased rate of cancer and life-threatening infections (Gallagher et al., JAm Soc Nephrol. 21:852-. 8 (2010)). In the context of ES cells, these drugs showed only a slight improvement in allowing survival across the MHC barrier (Swijnenburg et al., Proc Natl Acad Sci USA 105.12991-6( 2008); Toriumi et al., Neurol Res. 31:220-7 (2009)). Newer, more targeted immunosuppressive agents have become available, and skin and heart (Larsen et al., Nature. 381:434-8 (1996)) and ES cell allograft settings (Pearl et al., Cell Stem). Cell. 8:309-17 (2011)), they are still systemic and therefore likely to leave compromised host immunity.

One proposed advantage to the discovery of iPS cells was that they could be generated from and for each patient. These cells theoretically should be protected from immune rejection by the corresponding patients (Pearl et al., Sci Trans Med. 4: 164 ps25 (2012)). However, induction of iPS cell status involves epigenetic changes and in vitro culture pressures that can give rise to abnormal and malignant tumors, thus necessitating vigorous testing and/or genetic modification of each cell line to achieve safety and function. (Hussein et al., Nature. 471:58-62 (2011); Laurent et al., Cell Stem Cell. 8:106-18 (2011); Lister et al., Nature. 471:68-73 ( 2011)). Finally, the cost and time required to generate and test iPS cell lines for each individual patient make this approach practical and economically impractical. Even if the costs are dramatically reduced, it cannot help patients in need of immediate treatment due to conditions such as burns, heart attacks, strokes and spinal cord injuries (among others). Moreover, given recent findings, it is arguable whether iPS cell-derived cell types are truly protected from immune rejection even when transplanted into the same host from which they were derived (Zhao et al. al., Nature.474:212-5 (2011)).

One solution proposed in this regard is to use naturally suppressing or modulating immune cells, such as Tregs, which are proliferated and/or transplanted before, during or after transplantation of therapeutic cells or tissues. (Cobbold et al., Cold Spring Harb Perspect Med. 3(6) (2013); Wood et al., Nature reviews Immunology. 12: 417-30 (2012)). These strategies have led to the discovery of the master regulator FoxP3 that programs a subset of CD4+ cell regulatory T cells, specifically recognition of suppressive immune pathways (Hori et al., 299:1057-61 (2003); Fontenot et al., Nat). Immunol. 4: 330-6 (2003)) and proof of their critical importance in promoting resistance to allografts (Kendal et al., J Exp Med. 208:2043-53 (2011)). Is suggested based on. This idea contrasts with some of the first tolerization strategies that focused almost exclusively on depletion of effector T cells and creation of donor chimerism by monoclonal antibodies coupled to bone marrow transplants (Cobbold et al., Nature). 323:164-6 (1986); Qin et al., J Exp Med. 169:779-94 (1989)). The importance of suppressive T cell phenotype is that it does not kill cells, but leaves them unresponsive to allografts (Cobbold et al., J Immunol. 172:6003-10 (2004)), while at the same time others. Was evaluated later in a strategy that blocked key T cell receptors in a way that could suppress naive T cells of specificity (Cobbold et al., Immunol Rev. 129:165-201 (1992). Qin et al., Eur J Immunol. 20:2737-45 (1990)). These cells, which are currently recognized as Tregs, are TGFβ (Nakamura et al., The Journal of experimental medicine. 194:629-44 (2001); Nakamura et al., J Immunol. 172:834-42). ), CTLA4 (Tang et al., J Immunol. 181:1806-13 (2008); Walker et al., Trends Immunol. 36:63-70 (2015)), IL10 (O'Garra et al., J Clin. Invest. 114: 1372-8 (2004); Chaudhry et al., Immunity. 34:566-78 (2011)) and IL35 (Collison et al., Nature. 450:566-9 (2007)). Factor expression as well as preferential consumption of IL-2 (Shevach et al., Immunity. 30:636-45 (2009); Setoguchi et al., J Exp Med. 201:723-35 (2005)), antigen presentation. Manipulation or killing of cells (Mahnke et al., Cell Immunol. 250:1-13 (2007); Shevach et al., Immunol Rev. 212:60-73 (2006)) and local ATP (Regateiro et al.,). Eur J Immunol. 41: 2955-65 (2011); Regateiro et al., Clin Exp Immunol. 171: 1-7 (2013)) or an essential amino acid (Cobbold et al., Proc Natl Acad Sci U S A. 106:). Tolerance may be promoted by many mechanisms, including (but not limited to) depletion of 12055-60 (2009).

Two approaches for potential therapeutic use of Tregs are either in vitro expansion using donor antigen coupled with transplantation or selective in vivo expansion that takes advantage of the difference between regulatory T cells and effector T cells. Including or. These strategies are interesting, but to date, long-term acceptance of allografts has not been demonstrated solely by the use of expanded Tregs in vitro or in vivo. Many complications and unknown aspects remain in Treg biology, including optimal methodologies for in vitro or in vivo expansion and therapeutically relevant doses and timing. Moreover, antigen-specific Treg suppression can be “defeated” depending on the inflammatory situation (Korn et al., Nat Med. 13:423-31 (2007)), and Treg can be killed by NK cells. (Roy et al., J Immunol. 180:1729-36 (2008)).

In addition to Tregs, other suppressor cell types such as antigen presenting cells such as dendritic cells (DC) have also been explored to induce allograft resistance (Walker et al., Trends Immunol. 36:63. -70 (2015)). DCs are a link between innate and adaptive immunity and can induce both effector and suppressive immune responses depending on their maturation status and conditions such as local inflammatory cues. During allograft rejection, DC are co-stimulatory molecules such as CD80, CD86 and CD40 (among others) that are recognized and activated by allograft-specific T cell clones, along with MHC (mouse) or HLA (human). ) Presents allograft antigens inside the binding groove of the molecule on their surface (Walker et al., Trends Immunol. 36:63-70 (2015)). Tolerogenic DCs maintain predominantly low levels of MHC and costimulatory molecule expression and are then premature by exposure to repressive cues that promote naive T cells to anergic or even T regulatory subtypes during DC-T cell interactions. Can be derived from the condition.

Therapeutically, one application of this biology is to expand DCs that are simultaneously exposed in vitro to a specific allograft antigen and immunosuppressive factor of interest, many of which have been tested and Is TGF-β, IL10, cAMP, prostaglandin E2, histamine, neuropeptide, vitamin D2, B2 agonist, HLA-G, glucosamine and corticosteroids, cyclosporine, tacrolimus, rapamycin, aspirin, mecophenolate mofetil, san. Including drugs such as glyferin and deoxyspergualin (Hackstein et al., Nat Rev Immunol. 4:24-34 (2004)). Instead, DC directly target immunomodulators such as siRNA-induced silencing of TGF-β, IL-10, VEGF, FasL, CTLA4-Ig, IDO, NFKb decoy receptor, soluble TNFR, CCR7 and IL-12. It has been genetically engineered to express (Morelli et al., Immunol Rev. 196:125-46 (2003)). The hypothesis is that these cultured or engineered DCs are then transferred to the recipient at the same time as the allograft to suppress allograft-specific T cells or increase the number of allograft-concentrating T regulatory cells. Below, it is tested whether it can prolong the survival of allografts.

In one prototypical approach of this kind, bone marrow-derived DCs were transduced with SOCS1 (preventing up-regulation of costimulatory molecules and MHCII), which extended mouse heart allografts (Fu et al., Cell Mol Immunol. 6:87-95 (2009)). In another demonstration, FasL-expressing DCs were also able to prolong mouse heart allografts (Min et al., J Immunol. 164:161-7 (2000)). In general, there are many specific and combinatorial approaches that use tolerogenic DCs along these lineages (Bjorck et al., J Heart Lung Transplant. 24:1118-20 (2005): Sun et al.,. PLoS One.7:e52096 (2012); Li et al., J Immunol. 178:5480-7 (2007); Xu et al., Transplant Proc. 38:1561-3 (2006); Lan et al., J. Immunol. 177:5868-77 (2006); Lutz et al., Eur J Immunol. 30: 1813-22 (2000); Fischer et al., Transpl Immunol. 25:20-6 (2011)). Very variable depending on the type of DC modification, culture conditions, timing and type of allograft tested (Zhou et al., J Immunol Res. 2016: 5730674 (2016); Xia et al., J Evid Based Med. 7:135-46 (2014)). Almost all of these studies have been done in mice, but recent human studies have shown that safety studies in healthy volunteers (Dhodapkar et al., J Exp Med. 193:233-8 (2001); Dhodapkar et al., Blood. 100:174-7 (2002)) and a phase I clinical trial (Giannoukakis et al., Diabetes Care. 34:2026-32 (2011)) in 10 diabetic patients. Has been started.

Much remains unknown for both adoptive Treg and tolerogenic DC therapies, and one of the most important is the duration of their efficacy. In vivo studies have shown that using these two approaches (with or without additional immunosuppressive drugs), extended allograft survival is possible, but in the long term. None, and in almost all cases, the allograft eventually dies. This is in line with the fact that both Treg and DC have finite time spans. It is also possible to “transduce” and instead promote inflammatory pathways between tolerogenic phenotypes, especially DC (Delamarre et al., Semin Immunol. 23:2-11 (2011); Schreibelt et. al., Cancer Immunol Immunoother. 59: 1573-82 (2010); Satpathy et al., Nat Immunol. 14: 937-48 (2013)). This is likely due to the highly adaptive nature of DCs and their ability to sense and respond to a wide range of inflammatory cues. It has also been shown that these cells can die very quickly after adoptive transfer in vivo. Also, many subsets of suppressive Tregs and tolerogenic DCs have been described, which subtype is the ideal subtype, or whether it depends entirely on the context of allograft transplantation. Is still unknown.

Moreover, these types of approaches are of great practical and economic value in that they require the clinician to manipulate and work with complex immune cell types in addition to being therapeutic. There are barriers. Given their finite lifespan, it remains unclear whether these cells need to be delivered continuously and/or repeatedly to confer long-term tolerance to allografts. This complicates already expensive and timely methodologies for culturing, growing or transducing cells with key immunomodulators and ultimately prevents their use for very time-sensitive treatments.

Another approach to induce tolerance is the use of hematopoietic cell transplantation (HCT), where recipients of HLA mismatch organs undergo HCT using hematopoietic cells from the same donor (Gozzo et al. 21:281-4 (1970); Ildstad et al., Nature. 307:168-70 (1984); Sayegh et al., Ann Intern Med. 114:954-5 (1991); Huang et. al., J Clin Invest. 105:173-81 (2000); Kawai et al., N Engl J Med. 358:353-61 (2008); Sachs et al., Semin Immunol. 23:165-73 (2011). )). This results in a chimerism that can allow newly developing T and B cells in the recipient to be tolerant to both recipient and donor antigens (Tomita et al., J Immunol. 153:1087-98. (1994); Tomita et al., Transplantation. 61:469-77 (1996); Tomita et al., Transplantation. 61:477-85 (1996); Khan et al., Transplantation. 62:380-7 (1996). ); Manila et al., Transplantation. 66:96-102 (1998)). This is due to the role that hematopoietic cells play in positive and negative selection in the thymus, where they eliminate cells that have an affinity for hematopoietic cell-containing antigens that may also be present in allografts, and Lead to their rejection. (Griesemer et al, Transplantation. 90:465-74 (2010)). However, an inherent and dangerous risk of this approach is the potential for graft-versus-host disease (GVHD) in which the transplanted hematopoietic cells recognize the recipient tissue as foreign and systemically attack it (Sun et al. , PLoS One. 7:e52096 (2012)). Since its inception, several variants of HCT have been developed to suppress rejection, including the use of non-myeloablative strategies. These strategies use modified chemotherapeutic regimens, often with lower dosages, so that recipients undergoing HCT do not undergo complete elimination of their hematopoietic compartments. For example, recent ones of these strategies have used tolerance-promoting cell (FC)-based HCTs to largely avoid GHVD, while promoting tolerance in HLA-mismatched renal recipients (Leventhal et al. , Sci Transl Med. 4:124ra28 (2012)).

In addition to the risk of GHVD and the risk of secondary HCT procedures, a common limitation to these chimerism-inducing approaches is the need to have available HLA-matched donors for bone marrow harvest. This is easily achieved in rodent studies, but is very demanding in humans. The donor organ should ideally be harvested from the donor as soon as possible, which leaves an incredibly short window for harvesting bone marrow, if possible. It is also an expensive and logistically demanding procedure that requires a very patient and surgery specific approach. Also, as with the regulatory cell approach, it is not clear how the patient will benefit from the treatment of acute injuries or illnesses, or indeed how it will be applied in situations where immediate treatment is required.

Another approach that has been tested to reduce allo-rejection in vivo is the removal of histocompatibility molecules (Torikai et al., Blood. 122:1341-9 (2013)), which is associated with allo-rejection. It is the major source of antigenicity for "non-self" recognition. This fits the empirical data that HLA-matched donors and recipients significantly improved organ survival after transplantation (Opelz et al., Rev Immunogenet. 1:334-42 (1999)). Although there were some positive results with this approach, removal of MHC class I renders cells highly sensitive to NK cells (Pegram et al., Immunol Cell Biol. 89:216-24 (2011). Raulet et al., Nat Rev Immunol. 6:520-31 (2006); Huntington, Immunol Cell Biol. 92:208-9 (2014)). This approach also leaves the MHC-independent killing pathway between CD8+ T cells intact (Haspot et al., Am J Transplant. 14:49-58 (2014)) and also for antigens other than the MHC/HLA gene family. Difference (minor antigen) (Roopenian et al., Immunol Rev. 190:86-94 (2002)).

Uses of this approach included the deletion of all classical HLA class I molecules from pluripotent stem cells in combination with the introduction of a gene encoding HLA-E, the minimal polymorphic HLA that inhibits NK cells. (Gornalusse et al., Nat Biotechnol. (2017)). This approach demonstrated short-term resistance to NK and CD8 T cell challenge in partially immunodeficient humanized mice, but it was demonstrated that these cells could survive for long periods in a fully immunocompetent host. There wasn't. In another approach, ES cells were engineered to express PD-L1 and CTLA4-Ig, which improved survival in cognate hosts (Rong et al., Cell Stem Cell. 14:121-30. (2014)), with the severe limitation that CTLA4-Ig can lead to systemic immunosuppression. It has not yet been demonstrated that a series of modifications to ES cells or iPS cells allow them to escape allo-rejection without the possibility of systemic immunosuppression and without the need for immunosuppressive drugs.

The purpose of the present disclosure is to mitigate and/or eliminate one or more of the above drawbacks.

In one aspect, cells genetically modified to include at least one mechanism for providing local immunosuppression at the site of transplantation when transplanted into an allogeneic host are provided. A genetically modified cell is a set of transgenes, each transgene being a gene product that is cytosolic, membrane bound or locally acting, with the following functions: a) activation and function of antigen presenting cells. B) alleviating graft-attacking leukocyte activity or cytolytic function, c) alleviating macrophage cytolytic function and phagocytosis of allograft cells, and d) inducing apoptosis in graft-attacking leukocytes. A gene product having one or more of: e) mitigating local inflammatory proteins, and f) protecting against leukocyte-mediated apoptosis.

In a cell embodiment, the set of transgenes comprises the following genes: PD-L1, HLA-G (or mouse version of HLA-G, H2-M3), Cd47, Cd200, FASLG (or mouse version of FASLG, FasL. ), Ccl21 (or mouse version of Ccl21, Ccl21b), Mfge8 and Serpin B9 (or mouse version of Serpin B9, Spi6) one or more (eg 1, 2, 3, 4, 5, 5; 6, 7, or all 8).

In a cell embodiment, the set of transgenes comprises the following genes: PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6). Including two or more of.

In a cell embodiment, the set of transgene genes is PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6) or PD-. It includes genes encoding biological agents that act as agonists of L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6).

In a cell embodiment, the cell has the following genes: TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1 and IFNγR1 d39 or TGF-β, Cd73, Cd39, Lag3, Lag3, Lag3, Lag3, Lag3, Irg2, Irg2, Irg2, Irg3, Iag2, Iag3, Iag3, Iag3, Iag3, Iag3, Iag3, Iag2, Iag3, Id3, Id3, Id3, Id3, Id3, Id3, Id3, Id3, Id3, Id2. One or more of the genes encoding biological agents that act as agonists of Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1 or IFNγR1 d39 (eg 1, 2, 3, 4, 5, 5, 6) , 7, 8, 9, 10, or all 11) are further included.

In cellular embodiments, TGF-β or biological agents act locally within the implant environment. In cellular embodiments, TGF-β or biological agents act locally within the graft environment with minimal systemic effects.

In various embodiments of cells, the cells are stem cells, cells that are subject to genome editing and/or a source of therapeutic cell types (eg, cells that can be differentiated into therapeutic cell types or cells of the desired target tissue). In various embodiments, the cells are embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells, hematopoietic stem cells, mesenchymal stem cells, endothelial stem cells, epithelial stem cells, adipose stem cells or progenitor cells, reproductive stem cells, lung stem cells or progenitor cells. , Mammary gland stem cells, olfactory adult stem cells, hair follicle stem cells, intestinal stem cells or progenitor cells, multipotent stem cells, amniotic stem cells, cord blood stem cells, neural stem cells or progenitor cells, adult stem cells, somatic stem cells, tissue-specific stem cells, totipotent Stem cells, fibroblasts, monocyte precursor cells, B cells, exocrine cells, pancreatic precursor cells, endocrine precursor cells, hepatoblasts, myoblasts, preadipocytes, hepatocytes, chondrocytes, smooth muscle cells, K562 human erythrocytes Leukemia cell line, bone cell, synovial cell, tendon cell, ligament cell, meniscus cell, adipocyte, dendritic cell, natural killer cell, skeletal muscle cell, cardiomyocyte, erythroid-megakaryocytic cell, eosinophil Sphere, macrophage, T cell, islet β cell, neuron, cardiomyocyte, blood cell, exocrine precursor cell, duct cell, acinar cell, α cell, β cell, δ cell, PP cell, cholangiocyte, white or brown adipocyte , Hormone-secreting cells, epidermal keratinocytes, epithelial cells, renal cells, germ cells, skeletal joint synovial cells, periosteal cells, perichondrocytes, chondrocytes, endothelial cells, pericardial cells, meningeal cells, keratinocyte precursor cells, Keratinocyte stem cells, pericytes, glial cells, ependymal cells, cells isolated from amnion or placental membrane, serosa cells, somatic cells or skin, heart, brain or spinal cord, liver, lung, kidney, pancreas, bladder, bone marrow, Cells derived from the spleen, intestine or stomach.

In a cell embodiment, the cell has at least one (eg, reduces the tumorigenicity of modified cells or reduces the growth of modified cells that have become tumorigenic) (eg, reduces the growth of modified cells that become tumorigenic). 1, 2, 3, or more) mechanisms are further genetically modified to include these mechanisms. Genetically modified cells include genetic modifications of one or more (eg, one, two, three or more) cell division loci (CDL), where the CDL is at one or more loci. Wherein the transcript is one or more loci expressed by a dividing cell (eg, all dividing cells including one or more of the immunosuppressive transgenes) and the genetic modification is a). CDL comprising an ALINK system comprising a DNA sequence encoding a negative selection marker transcriptionally linked to a DNA sequence encoding CDL, and b) an inducible activator-based gene expression system operably linked to CDL Is one or more of the exogenous activator (EARC) system of regulation.

In a cell embodiment, the genetic modification of CDL is carried out using one or more of a) a DNA vector containing an ALINK system, b) a DNA vector containing an EARC system, and c) a DNA vector containing an ALINK system and an EARC system. The ALINK and/or EARC systems are each operably linked to the CDL, which involves making targeted substitutions of the CDL.

In various embodiments of the cell, the ALINK gene modification of the CDL is homozygous, heterozygous, hemizygous or complex heterozygous, and/or the EARC gene modification is a functional CDL modification is an EARC modification allele. Ensure that it can only be produced through genes.

In various embodiments of the cell, the CDL is one or more of the loci listed in Table 5 (eg, 1, 2, 3, or more). In various embodiments, the CDL encodes a gene product that functions in one or more of the cell cycle, DNA replication, RNA transcription, protein translation and metabolism. In various embodiments, the CDL is one or more of Cdk1/CDK1, Top2A/TOP2A, Cenpa/CENPA, Birc5/BIRC5 and Eef2/EEF2, preferably the CDL is Cdk1 or CDK1. In some embodiments, the CDL is Top2A. In some embodiments, the CDL is Eef2. In various embodiments, the CDL is two or more of Cdk1/CDK1, Top2A/TOP2A, Cenpa/CENPA, Birc5/BIRC5 and Eef2/EEF2, preferably the CDL is Cdk1 and Top2A or Cdk1 and Eef2. ..

In various embodiments of the cells, the ALINK system comprises the herpes simplex virus-thymidine kinase/gancyclovir system, the cytosine deaminase/5-fluorocytosine system, the carboxylesterase/irinotecan system or the iCasp9/AP1903 system, preferably the ALINK system. , Herpes simplex virus-thymidine kinase/gancyclovir system.

In various embodiments of the cell, the EARC system is a doxycycline-induced "dox-crosslinking" system, a cumate switch-inducible system, an ecdysone-inducible system, a radio-induced system or a ligand reversible dimerization system, preferably the EARC system. Is a dox-crosslinked system.

In one aspect, methods are provided for providing local immunosuppression at a transplant site in a homologous host. The method comprises providing a cell and expressing a set of transgenes in the cell, each transgene being a gene product that is cytoplasmic, membrane bound or locally acting Functions: a) mitigating activation and function of antigen-presenting cells, b) mitigating graft-attacking leukocyte activity or cytolytic function, and c) mitigating macrophage cytolytic function and phagocytosis of allograft cells. , D) inducing apoptosis in graft-challenged leukocytes, e) mitigating local inflammatory proteins, and f) protecting against leukocyte-mediated apoptosis.

In an embodiment of the method, the set of transgenes comprises the following genes: PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6). ) Or PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 or Serpin B9 (Spi6) of a gene encoding a biological substance that acts as an agonist. One or more (eg, one, two, three, four, five, six, seven or all eight).

In an embodiment of the method, the set of transgenes comprises: ) Is included.

In an embodiment of the method, the set of transgene genes is PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6) or PD. -L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6).

In an embodiment of the method, the method is the following genes: TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1 and IFNganmaaru1 d39 or TGF-β, Cd73, Cd39, Lag3, Il1r2 , Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1 or IFNγR1 d39 one or more of the genes encoding biological agents that act as agonists (eg 1, 2, 3, 4, 5, 5, 6 One, seven, eight, nine, ten or all) are further included. In an embodiment of the method the TGF-β or biological agent acts locally within the implant environment. In embodiments, the TGF-β or biological agent acts locally within the implant environment with minimal systemic effects.

In various embodiments of the method, the cells are stem cells, cells that are subject to genome editing, and/or a source of therapeutic cell types (eg, cells that can be differentiated into therapeutic cell types or cells of the desired target tissue). In various embodiments, the cells are embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells, hematopoietic stem cells, mesenchymal stem cells, endothelial stem cells, epithelial stem cells, adipose stem cells or progenitor cells, reproductive stem cells, lung stem cells or progenitor cells. , Mammary gland stem cells, olfactory adult stem cells, hair follicle stem cells, intestinal stem cells or progenitor cells, multipotent stem cells, amniotic stem cells, cord blood stem cells, neural stem cells or progenitor cells, adult stem cells, somatic stem cells, tissue-specific stem cells, totipotent Stem cells, fibroblasts, monocyte precursor cells, B cells, exocrine cells, pancreatic precursor cells, endocrine precursor cells, hepatoblasts, myoblasts, preadipocytes, hepatocytes, chondrocytes, smooth muscle cells, K562 human erythrocytes Leukemia cell line, bone cell, synovial cell, tendon cell, ligament cell, meniscus cell, adipocyte, dendritic cell, natural killer cell, skeletal muscle cell, cardiomyocyte, erythroid-megakaryocytic cell, eosinophil Sphere, macrophage, T cell, islet β cell, neuron, cardiomyocyte, blood cell, exocrine precursor cell, duct cell, acinar cell, α cell, β cell, δ cell, PP cell, cholangiocyte, white or brown adipocyte , Hormone-secreting cells, epidermal keratinocytes, epithelial cells, renal cells, germ cells, skeletal joint synovial cells, periosteal cells, perichondrocytes, chondrocytes, endothelial cells, pericardial cells, meningeal cells, keratinocyte precursor cells, Keratinocyte stem cells, pericytes, glial cells, ependymal cells, cells isolated from amnion or placental membrane, serosa cells, somatic cells or skin, heart, brain or spinal cord, liver, lung, kidney, pancreas, bladder, bone marrow, Cells derived from the spleen, intestine or stomach.

In various embodiments of the method, cells are provided (eg, injected) at or near the site of implantation. In various embodiments of the method, the cells are provided (eg, injected or transplanted) within a graft (eg, injected or transplanted into a tissue or organ graft before, during, or after transplantation). ). In some embodiments, the transgene-expressing cells are transplanted cells (eg, transplanted tissue or organ cells are PD-L1, HLA-G(H2-M3), Cd47, Cd200. , FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6) one or more (eg 1, 2, 3, 4, 5, 5, 6, 7 or all 8) ) Is modified to express)).

In one aspect, a method of controlling the growth of cells at a transplant site in a cognate host is provided (eg, cells that reduce the tumorigenicity of cells at the transplant site or become tumorigenic at the transplant site). To reduce growth). The method comprises a) genetically modifying one or more (eg, one, two, three or more) cell division loci (CDL) in a cell, where the CDL is One or more loci, the transcript of which is one or more loci expressed by a dividing cell (eg, all dividing cells including one or more of the immunosuppressive transgenes). , A genetic modification of the CDL, i) an ablation link (ALLINK) system comprising a DNA sequence encoding a negative selection marker transcriptionally linked to the DNA sequence encoding the CDL, and i) induction operably linked to the CDL. Genetically modifying one or more of the inducible exogenous activator (EARC) systems of regulation of CDL, including sexual activator-based gene expression systems; b) local immunosuppression at the transplant site. Genetically modifying the cell to include at least one mechanism for providing, c) transplanting the cell or population of cells at a transplant site in a cognate host, and d) a genetically modified cell containing the ALINK system as a negative selectable marker. By maintaining it in the absence of an inducer of A. coli, or allowing the growth of genetically modified cells containing the ALINK system, or exposing the ALINK system-containing cells to the inducer of the negative selection marker. Genes containing the EARC system by ablating and/or inhibiting the growth of the gene-modified cells containing the ERC system and/or exposing the gene modified cells containing the EARC system to inducers of gene expression systems based on inducible activators By allowing the growth of modified cells, or maintaining cells containing the EARC system in the absence of inducers of gene expression systems based on inducible activators, the growth of genetically modified cells containing the EARC system is increased. Including prevention or inhibition.

In an embodiment of the method, the CDL gene is modified by using one or more of a) a DNA vector containing an ALINK system, b) a DNA vector containing an EARC system, and c) a DNA vector containing an ALINK system and an EARC system. And making targeted substitutions of the CDL, wherein the ALINK and/or EARC systems are each operably linked to the CDL.

In various embodiments of the method, the ALINK gene modification of the CDL is homozygous, heterozygous, hemizygous or complex heterozygous, and/or the EARC gene modification is a functional CDL modification is an EARC modification allele. Ensure that it can only be produced through genes.

In various embodiments of the method, the CDL is one or more of the loci listed in Table 5 (eg, 1, 2, 3, or more). In various embodiments, the CDL encodes a gene product whose function is involved in one or more of cell cycle, DNA replication, RNA transcription, protein translation and metabolism. In various embodiments, the CDL is one or more of Cdk1/CDK1, Top2A/TOP2A, Cenpa/CENPA, Birc5/BIRC5 and Eef2/EEF2, preferably the CDL is Cdk1 or CDK1. In some embodiments, the CDL is Top2A. In some embodiments, the CDL is Eef2. In various embodiments, the CDL is two or more of Cdk1/CDK1, Top2A/TOP2A, Cenpa/CENPA, Birc5/BIRC5 and Eef2/EEF2, preferably the CDL is Cdk1 and Top2A or Cdk1 and Eef2. ..

In various embodiments of the method, the ALINK system comprises the herpes simplex virus-thymidine kinase/gancyclovir system, the cytosine deaminase/5-fluorocytosine system, the carboxylesterase/irinotecan system or the iCasp9/AP1903 system, preferably the ALINK system. , Herpes simplex virus-thymidine kinase/gancyclovir system.

In various embodiments of the method, the EARC system is a doxycycline-induced “dox-bridge” system, a cumate switch-inducible system, an ecdysone-inducible system, a radio-induced system or a ligand reversible dimerization system, preferably the EARC system. Is a dox-crosslinked system.

In an embodiment of the method, the genetically modified cells are a set of transgenes, each transgene being a gene product that is cytoplasmic, membrane bound or locally acting, with the following functions: a) antigen Alleviating the activation and function of the presentation cells, b) alleviating the graft-attacking leukocyte activity or cytolytic function, c) alleviating the macrophage cytolytic function and phagocytosis of allograft cells, d) the transplantation It includes a set of transgenes that encode gene products having one or more of inducing apoptosis in one-sided leukocytes, e) mitigating local inflammatory proteins, and f) protecting against leukocyte-mediated apoptosis.

In an embodiment of the method, the set of transgenes comprises the following genes: PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6). ) Or PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 or Serpin B9 (Spi6) of a gene encoding a biological substance that acts as an agonist. One or more (eg, one, two, three, four, five, six, seven or all eight).

In an embodiment of the method, the set of transgenes comprises: ) Is included.

In an embodiment of the method, the set of transgene genes is PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6) or PD. -L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6).

In an embodiment of the method, the cell, the following genes: TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1 and IFNganmaaru1 d39 or TGF-β, Cd73, Cd39, Lag3, Il1r2 , Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1 or IFNγR1 d39 one or more of the genes encoding biological agents that act as agonists (eg 1, 2, 3, 4, 5, 5, 6 1, 7, 8, 9, 10, or all 11) are further included.

In an embodiment of the method the TGF-β or biological agent acts locally within the implant environment. In embodiments, the TGF-β or biological agent acts locally within the implant environment with minimal systemic effects.

In various embodiments of the method, the cells are stem cells, cells that are subject to genome editing, and/or a source of therapeutic cell types (eg, cells that can be differentiated into therapeutic cell types or cells of the desired target tissue). In various embodiments, the cells are embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells, hematopoietic stem cells, mesenchymal stem cells, endothelial stem cells, epithelial stem cells, adipose stem cells or progenitor cells, reproductive stem cells, lung stem cells or progenitor cells. , Mammary gland stem cells, olfactory adult stem cells, hair follicle stem cells, intestinal stem cells or progenitor cells, multipotent stem cells, amniotic stem cells, cord blood stem cells, neural stem cells or progenitor cells, adult stem cells, somatic stem cells, tissue-specific stem cells, totipotent Stem cells, fibroblasts, monocyte precursor cells, B cells, exocrine cells, pancreatic precursor cells, endocrine precursor cells, hepatoblasts, myoblasts, preadipocytes, hepatocytes, chondrocytes, smooth muscle cells, K562 human erythrocytes Leukemia cell line, bone cell, synovial cell, tendon cell, ligament cell, meniscus cell, adipocyte, dendritic cell, natural killer cell, skeletal muscle cell, cardiomyocyte, erythroid-megakaryocytic cell, eosinophil Sphere, macrophage, T cell, islet β cell, neuron, cardiomyocyte, blood cell, exocrine precursor cell, duct cell, acinar cell, α cell, β cell, δ cell, PP cell, cholangiocyte, white or brown adipocyte , Hormone-secreting cells, epidermal keratinocytes, epithelial cells, renal cells, germ cells, skeletal joint synovial cells, periosteal cells, perichondrocytes, chondrocytes, endothelial cells, pericardial cells, meningeal cells, keratinocyte precursor cells, Keratinocyte stem cells, pericytes, glial cells, ependymal cells, cells isolated from amnion or placental membrane, serosa cells, somatic cells or skin, heart, brain or spinal cord, liver, lung, kidney, pancreas, bladder, bone marrow, Cells derived from the spleen, intestine or stomach.

In various embodiments of the method, cells are provided (eg, injected) at or near the site of implantation. In various embodiments of the method, the cells are provided (eg, injected or transplanted) within a graft (eg, injected or transplanted into a tissue or organ graft before, during, or after transplantation). ). In some embodiments, the transgene-expressing cells are transplanted cells (eg, transplanted tissue or organ cells are PD-L1, HLA-G(H2-M3), Cd47, Cd200. , FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6) one or more (eg 1, 2, 3, 4, 5, 5, 6, 7 or all 8) ) Is modified to express)).

In various embodiments of the method, the homologous host is a mammal. In various embodiments of the method, the cognate host is a mouse or human.

In various embodiments of the method, the host has a degenerative disease or condition that can be treated with cell therapy. In various embodiments, the disease or condition is blindness, arthritis (eg, osteoarthritis or rheumatoid arthritis), ischemia, diabetes (eg, type 1 or type 2 diabetes), multiple sclerosis, spinal cord injury, stroke. , Cancer, lung disease, blood disease, Parkinson's disease, Alzheimer's disease, Huntington's disease and neurological diseases such as ALS, enzyme or hormone deficiency, metabolic disorder (eg, lysosomal storage disorder, galactoseemia, maple syrup urine disease, phenylketonuria) Disease, glycogen storage disease, mitochondrial disorder, Friedrich's ataxia, peroxisome disorder, metal metabolism disorder or organic acidemia), autoimmune disease (eg, psoriasis, systemic lupus erythematosus, Grave's disease, inflammatory bowel disease, Addison's disease, Sjogren's syndrome, Hashimoto thyroiditis, vasculitis, autoimmune hepatitis, alopecia areata, autoimmune pancreatitis, Crohn's disease, ulcerative colitis, dermatomyositis), age-related macular degeneration, retinal dystrophy, infection, hemophilia Disease, degenerative disease (eg, Charcot-Marie-Tooth disease, chronic obstructive pulmonary disease, chronic traumatic encephalopathy, Creutzfeld-Jakob disease, cystic fibrosis, cytochrome c oxidase deficiency, Ehrers-Danlos syndrome, essential Tremor, progressive ossifying fibrodysplasia, infantile axonal dystrophy, keratoconus, spherical cornea, muscular dystrophy, neuronal ceroid lipofuscinosis, previous disease, progressive supranuclear palsy, Sandhoff disease , Spinal muscular atrophy, retinitis pigmentosa) or age-related diseases (eg, atherosclerosis, cardiovascular disease), cardiovascular disease (eg, angina pectoris, myocardial infarction), cataract, osteoporosis or hypertension ).

In some embodiments of any of the above aspects, 1 of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6). One or more (eg 1, 2, 3, 4, 5, 5, 6, 7 or all 8) are expressed at or above the level of expression of the corresponding endogenous gene in activated leukocytes. (Eg, the expression level of the T cell, eg, the cloaking transgene, is equal to the expression level of the endogenous gene in activated leukocytes, or 1.5, 2, than the expression level of the endogenous gene in activated leukocytes, 3, 4, 5, 6, 7, 8, 9, 10 times higher). In some embodiments, all eight of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6) are activated leukocytes. Is expressed at a level equal to or higher than the expression level of the corresponding endogenous gene in.

In some embodiments of any of the above aspects, 1 of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6). One or more (eg, one, two, three, four, five, six, seven or all eight) expressed at a level above that of the corresponding endogenous gene in wild-type stem cells. (Eg, the expression level of a wild-type ES cell from the same species, eg, a cloaking transgene, is at least a few 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 500, 1,000 times higher). In some embodiments, all eight of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6) are wild type stem cells. Higher than the expression level of the endogenous gene (eg, embryonic stem cells from the same species as cloak cells) (eg, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40) , 50 or 100 times higher). In some embodiments, one or more of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6) (eg, 1 One, two, three, four, five, six, seven or all eight) are expressed at a level in the top 5% of gene expression for all genes in the ES cell genome. In some embodiments, one or more of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6) (eg, 1 One, two, three, four, five, six, seven or all eight) are expressed at a level in the top 1% of gene expression for all genes in the ES cell genome. In some embodiments, PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6) are all in the ES cell genome. It is expressed at levels that are in the top 5% of gene expression for all genes.

In some embodiments of any of the above aspects, the PD-L1 transgene has at least 85% identity (eg, 85%, 90%, 95) to the amino acid sequence of SEQ ID NO:11 or SEQ ID NO:12. %, 96%, 97%, 98%, 99% or more of sequence identity).

In some embodiments of any of the above aspects, the HLA-G(H2-M3) transgene has at least 85% identity (eg, 85% identity) to the amino acid sequence of SEQ ID NO:16 or SEQ ID NO:15. , 90%, 95%, 96%, 97%, 98%, 99% or more).

In some embodiments of any of the above aspects, the Cd47 transgene has at least 85% identity to the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4 (eg, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity).

In some embodiments of any of the aforementioned aspects, the CD200 transgene has at least 85% identity to the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6 (eg, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity).

In some embodiments of any of the aforementioned aspects, the FASLG(FasL) transgene has at least 85% identity (eg, 85%, 90%, to the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:9). Encoding proteins with 95%, 96%, 97%, 98%, 99% or greater sequence identity).

In some embodiments of any of the above aspects, the Ccl21 (Ccl21b) transgene has at least 85% identity (eg, 85%, 90%, to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:1). Encoding proteins with 95%, 96%, 97%, 98%, 99% or greater sequence identity).

In some embodiments of any of the aforementioned aspects, the Mfge8 transgene has at least 85% identity to the amino acid sequence of SEQ ID NO:13 or SEQ ID NO:14 (eg, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity).

In some embodiments of any of the above aspects, the Serpin B9 (Spi6) transgene has at least 85% identity (eg, 85%, 90%) to the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:7. , 95%, 96%, 97%, 98%, 99% or more of sequence identity).

In some embodiments of any of the above aspects, the IFNγR1 d39 transgene has at least 85% identity to the amino acid sequence of SEQ ID NO:17 (eg, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity).

In some embodiments of any of the above aspects, the one or more transgenes are operably linked to a constitutive promoter. In some embodiments, the constitutive promoter is a CAG promoter, cytomegalovirus (CMV) promoter, EF1α promoter, PGK promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, HSV tk promoter, mouse. It is selected from the group consisting of the mammary tumor virus (MMTV) promoter, the HIV LTR promoter, the Moloney virus promoter, the Epstein Barr virus (EBV) promoter and the Rous sarcoma virus (RSV) promoter.

In some embodiments of any of the aforementioned aspects, the cell further comprises a transgene encoding a therapeutic agent (eg, the cell is modified to further comprise a transgene encoding a therapeutic agent). In some embodiments, the therapeutic agent is a protein or antibody. In some embodiments, the antibody is an inhibitory antibody or an agonist antibody. In some embodiments, the therapeutic agent is an agent listed in Table 2. In some embodiments, the therapeutic agent is a wild-type version of a gene that is mutated in the subject (eg, a wild-type version of a mutated gene associated with a disease or condition in the subject, such as cancer, enzyme or hormone deficiency, metabolic disorder). Or a genetic mutation associated with a degenerative disease). In some embodiments, the therapeutic agent is from the group consisting of a tetracycline response element, a light inducible system, a radiogenic system, a cumate switch inducible system, an ecdysone inducible system, a destabilization domain system or a ligand reversible dimerization system. Expressed using the inducible expression system of choice. In some embodiments, the therapeutic agent is a CAG promoter, cytomegalovirus (CMV) promoter, EF1α promoter, PGK promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, HSV tk promoter, mouse breast. It is expressed using a constitutive promoter selected from the group consisting of the tumor virus (MMTV) promoter, the HIV LTR promoter, the Moloney virus promoter, the Epstein Barr virus (EBV) promoter and the Rous sarcoma virus (RSV) promoter.

In another aspect, a population of genetically modified cells according to any of the above cells is provided.

In one aspect, methods are provided for providing local immunosuppression at a transplant site in a homologous host. The method comprises transplanting a genetically modified cell as described above or a population of genetically modified cells as described above at a transplant site in a cognate host.

In another aspect, the invention features a composition that includes a cell of the invention. In some embodiments, the composition further comprises a pharmaceutically acceptable excipient.

Another aspect features a kit comprising the cells of the invention or the pharmaceutical composition of the invention.

Another aspect features a method of treating a disease or condition in a subject in need thereof by administering the cells of the invention or the composition of the invention to the subject. In some embodiments, the disease or condition is blindness, arthritis (eg, osteoarthritis or rheumatoid arthritis), ischemia, diabetes (eg, type 1 or type 2 diabetes), multiple sclerosis, spinal cord injury, Stroke, cancer, lung disease, blood disease, neurological disease such as Parkinson's disease, Alzheimer's disease, Huntington's disease and ALS, enzyme or hormone deficiency, metabolic disorder (eg lysosomal storage disorder, galactoseemia, maple syrup urine disease, phenylketone) Urine, glycogen storage disease, mitochondrial disorder, Friedrich's ataxia, peroxisome disorder, metal metabolism disorder or organic acidemia, autoimmune disease (eg, psoriasis, systemic lupus erythematosus, Grave's disease, inflammatory bowel disease, Addison's disease) , Sjogren's syndrome, Hashimoto thyroiditis, vasculitis, autoimmune hepatitis, alopecia areata, autoimmune pancreatitis, Crohn's disease, ulcerative colitis, dermatomyositis), age-related macular degeneration, retinal dystrophy, infection, blood Friendship, degenerative diseases (eg Charcot-Marie-Tooth disease, chronic obstructive pulmonary disease, chronic traumatic encephalopathy, Creutzfeld-Jakob disease, cystic fibrosis, cytochrome c oxidase deficiency, Ehrers-Danlos syndrome, essential condition) Tremor, progressive ossifying fibrodysplasia, infantile neuroaxonal dystrophy, keratoconus, spheroidal cornea, muscular dystrophy, neuronal ceroid lipofuscinosis, previous disease, progressive supranuclear palsy, Sandhoff disease, spinal muscle Atrophy, retinitis pigmentosa) or age-related diseases (eg, atherosclerosis, cardiovascular diseases (eg, angina, myocardial infarction), cataracts, osteoporosis or hypertension) or diseases listed in Table 2. Or a wild-type version of a gene that has been mutated in the subject and/or the condition (eg, associated with a wild-type version of a mutated gene associated with the disease or condition in the subject, eg, cancer, enzyme or hormone deficiency, metabolic disorder or degenerative disease). Gene mutation). In some embodiments, the disease or condition is age-related macular degeneration (eg, wet AMD) or retinal dystrophy and the therapeutic agent is a VEGF inhibitor (eg, a soluble form of the VEG receptor (eg, Soluble VEGFR-1 or NRP-1), platelet factor-4, prolactin, SPARC, VEGF inhibitory antibody (eg bevacizumab or ranibizumab) or Holash et al., Proc Natl Acad Sci U.S.A. 99: 11383-. 11398, 2002, for example, soluble decoy receptors such as VEGF-Trap _parental , VEGF-Trap _ΔB1 , VEGF-Trap _ΔB2 , VEGF-Trap _R1R2 , eg afliberept. In some embodiments, the disease or condition is osteoarthritis or rheumatoid arthritis and the therapeutic agent is an anti-inflammatory biological agent (eg, a TNFα inhibitor (eg, adalimumab, etanercept, infliximab, golimumab). Or sertolizumab), an interleukin-6 (IL6) receptor inhibitor (eg tocilizumab), an IL1 receptor inhibitor (eg anakinra) or another agent used to treat rheumatoid arthritis (eg abatacept, Rituximab)). In some embodiments, the disease or condition is diabetes (eg, type 1 diabetes or type 2 diabetes) and the therapeutic agent is insulin. In some embodiments, the disease or condition is hemophilia and the therapeutic agent is Factor VIII. In some embodiments, the disease or condition is a metabolic deficiency and the therapeutic agent encodes a transgene having a nucleic acid sequence of the wild-type version of the gene that is mutated in the subject or an enzyme that is deficient in the subject. The transgene.

In some embodiments of any of the above aspects, the cells are differentiated into a lineage restricted cell type prior to administration to the subject. In some embodiments, the disease or condition is myocardial infarction and the cells are differentiated into cardiomyocytes. In some embodiments, the disease or condition is blind and the cells are differentiated into photoreceptor cells. In some embodiments, the disease or condition is spinal cord injury, Parkinson's disease, Huntington's disease or Alzheimer's disease, and the cells are dissociated into neurons. In some embodiments, the disease or condition is multiple sclerosis and the cells are differentiated into glial cells.

In some embodiments of any of the above aspects, the cells are locally administered (eg, infused or transplanted) to a tissue or body site in need of the cells or therapeutic agent.

In some embodiments of any of the above aspects, the cells are administered intravenously, subcutaneously, intramuscularly, transdermally, intradermally, parenterally, intraarterially, intravascularly or by perfusion.

In some embodiments of any of the foregoing aspects, the cells are administered by subcutaneous injection to produce cloaked subcutaneous tissue.

In some embodiments of any of the above aspects, the cells are administered as a tissue. In some embodiments, the tissue is administered with a gel, biocompatible matrix or cell scaffold.

In some embodiments of any of the aforementioned aspects, the cells are from 25,000 to 5,000,000 cells (eg, 2.5×10 ⁴ , 5×10 ⁴ , 7.5×). 10 ⁴ , 1×10 ⁵ , 2×10 ⁵ , 3×10 ⁵ , 4×10 ⁵ , 6×10 ⁵ , 6×10 ⁵ , 7×10 ⁵ , 8×10 ⁵ , 9×10 ⁵ , 1× 10 ⁶ , 2×10 ⁶ , 3×10 ⁶ , 4×10 ⁶ , 5×10 ⁶ , 6×10 ⁶ , 7×10 ⁶ , 8×10 ⁶ , 9×10 ⁶ , 1×10 ⁷ , 2× 10 ⁷ , 3×10 ⁷ , 4×10 ⁷ , 5×10 ⁷ , 6×10 ⁷ , 7×10 ⁷ , 8×10 ⁷ , 9×10 ⁷ , 1×10 ⁸ , 2×10 ⁸ , 3× 10 ⁸ , 4×10 ⁸ , 5×10 ⁸ , 6×10 ⁸ , 7×10 ⁸ , 8×10 ⁸ , 9×10 ⁸ , 1×10 ⁹ , 2×10 ⁹ , 3×10 ⁹ , 4× Doses of 10 ⁹ or 5×10 ⁹ cells).

In some embodiments of any of the above aspects, the cells are 800,000,000-100,000,000,000,000 cells (eg, 8×10 ⁸ , 9×10 ⁸ , 1×10 ^{9 ).} 2×10 ⁹ or 3×10 ⁹ , 4×10 ⁹ , 5×10 ⁹ , 6×10 ⁹ , 7×10 ⁹ , 8×10 ⁹ , 9×10 ⁹ , 1×10 ¹⁰ , 2×10 ^10. 3×10 ¹⁰ , 4×10 ¹⁰ , 5×10 ¹⁰ , 6×10 ¹⁰ , 7×10 ¹⁰ , 8×10 ¹⁰ , 9×10 ¹⁰ or 1×10 ¹¹ cells). ..

In some embodiments of any of the foregoing methods, the method further comprises administering an additional therapeutic agent. In some embodiments, the additional therapeutic agent is administered prior to administration of the cells. In some embodiments, the additional therapeutic agent is administered after administration of the cells. In some embodiments, the additional therapeutic agent is administered at the same time as the administration of the cells. In some embodiments, the additional therapeutic agent is an immunosuppressant agent, a disease modifying anti-rheumatic drug (DMARD), a biological response modifier (a type of DMARD), a corticosteroid or a nonsteroidal anti-inflammatory drug (NSAID). ), Prednisone, Prednisolone, Methylprednisolone, Methotrexate, Hydroxychloroquine, Sulfasalazine, Leflunomide, Cyclophosphamide, Azathioprine, Tofacitinib, Adalimumab, Abatacept, Anakinra, Kainelet, Certrizumab, Etanercept, Golixumab, Infrimab, Inflimab, Inflimab. Purine, 6-thioguanine, abatacept, adalimumab, alemtuzumab, aminosalicylate, antibiotics, antihistamine, anti-TNFα, azathioprine, belimumab, β-interferon, calcineurin inhibitor, certolizumab, corticosteroids, cromolyn, cyclosporin A, cyclosporin, fumarine. Dimethyl acid, etanercept, fingolimod, fumarate, glatiramer acetate, golimumab, hydroxyurea, IFNγ, IL-11, leflunomide, leukotriene receptor antagonist, long-acting β2 agonist, mitoxantrone, mycophenolate mofetil, Natalizumab, ocrelizumab, pimecrolimus, probiotic, retinoid, salicylic acid, short-acting β2 agonist, sulfasalazine, tacrolimus, teriflunomide, theophylline, tocilizumab, ustekinumab or vedolizumab, bevacuzumab, raniveluzumab, light-force, afrivelumab, afliberumato, afliberumato, afrivelumato, afliberumato Coagulation, carbidopa-levodopa, dopamine agonist, MAO-B inhibitor, catechol-O-methyltransferase inhibitor, anticholinergic, amantadine, deep brain stimulant, anticoagulant, antiplatelet agent, angiotensin converting enzyme inhibitor, Angiotensin II receptor blocker, Angiotensin receptor neprilysin inhibitor, β blocker, combined α and β blocker, calcium channel blocker, cholesterol lowering drug, nicotinic acid, cholesterol absorption inhibitor, digitalis preparation, diuretic, vasodilator Agent, antiplatelet dual therapy, cardiac treatment, antiviral compound, nucleoside analog reverse transcriptase inhibitor (NRTI), non-nucleoside reverse transcriptase inhibitor (NNRTI), protease inhibitor, antibacterial Compound, antifungal compound, antiparasitic compound, insulin, sulfonylurea, biguanide, meglitinide, thiazolidinedione, DPP-4 inhibitor, SGLT2 inhibitor, α-glucosidase inhibitor, bile acid sequestrant, aspirin, diet therapy, coagulation factor , Desmopressin, blood clot preservative, fibrin sealant, physiotherapy, coenzyme, bone marrow transplant, organ transplant, hemodialysis, hemofiltration, exchange transfusion, peritoneal dialysis, medium chain triacylglycerol, miglustat, enzyme replacement therapy, checkpoint Inhibitors, chemotherapeutic agents, biological agents, radiation therapy, cryotherapy, hyperthermia, surgical resection or tumor tissue or anti-cancer vaccines.

In some embodiments of any of the foregoing methods, the method further comprises controlling cell proliferation. In some embodiments, the cells comprise an ALINK system, and methods of controlling proliferation include i) maintaining the ALINK system-containing cells in the absence of an inducer of a negative selectable marker. Or ii) ablating or inhibiting the growth of cells containing the ALINK system by exposing the cells containing the ALINK system to an inducer of a negative selection marker. In some embodiments, the cell comprises an EARC system, and the method of controlling cell proliferation comprises: i) exposing the cell comprising the EARC system to an inducer of an inducible activator-based gene expression system. , EARC system by allowing the growth of cells containing the EARC system, or ii) maintaining cells containing the EARC system in the absence of inducers of gene expression systems based on inducible activators It includes preventing or inhibiting the growth of cells.

In some embodiments of any of the foregoing methods, the cells are removed after completion of treatment. Removal of cells can be by surgery (eg, to remove transplanted tissue or organs or to remove cloaked subcutaneous tissue) or by use of the ALINK and/or EARC system. In some embodiments, one or more (eg, one, two, three, four or more) ALINK and/or EARC lines are used to eliminate all of the cloak cells. ..

In another aspect, the invention provides a cell of the invention or a composition of the invention for use in treating a disease or condition in a subject in need thereof. In some embodiments, the disease or condition is blindness, arthritis (eg, osteoarthritis or rheumatoid arthritis), ischemia, diabetes (eg, type 1 or type 2 diabetes), multiple sclerosis, spinal cord injury, Stroke, cancer, lung disease, blood disease, neurological disease such as Parkinson's disease, Alzheimer's disease, Huntington's disease and ALS, enzyme or hormone deficiency, metabolic disorder (eg lysosomal storage disorder, galactoseemia, maple syrup urine disease, phenylketone) Urine, glycogen storage disease, mitochondrial disorder, Friedrich's ataxia, peroxisome disorder, metal metabolism disorder or organic acidemia, autoimmune disease (eg, psoriasis, systemic lupus erythematosus, Grave's disease, inflammatory bowel disease, Addison's disease) , Sjogren's syndrome, Hashimoto thyroiditis, vasculitis, autoimmune hepatitis, alopecia areata, autoimmune pancreatitis, Crohn's disease, ulcerative colitis, dermatomyositis), age-related macular degeneration, retinal dystrophy, infection, blood Friendship, degenerative diseases (eg Charcot-Marie-Tooth disease, chronic obstructive pulmonary disease, chronic traumatic encephalopathy, Creutzfeld-Jakob disease, cystic fibrosis, cytochrome c oxidase deficiency, Ehrers-Danlos syndrome, essential condition) Tremor, progressive ossifying fibrodysplasia, infantile neuroaxonal dystrophy, keratoconus, spheroidal cornea, muscular dystrophy, neuronal ceroid lipofuscinosis, previous disease, progressive supranuclear palsy, Sandhoff disease, spinal muscle Atrophy, retinitis pigmentosa) or age-related diseases (eg, atherosclerosis), cardiovascular diseases (eg, angina, myocardial infarction), cataracts, osteoporosis or hypertension) or listed in Table 2. A disease or condition.

In another aspect, the invention provides a cell of the invention or a composition of the invention for use in providing local immunosuppression at a transplant site in a cognate host.

In some embodiments of any of the aforementioned aspects, the cells are transgene PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9. Two of the (Spi6) sets (eg PD-L1 and HLA-G (H2-M3); PD-L1 and Cd47; PD-L1 and Cd200; PD-L1 and FASLG (FasL); PD-L1 and Ccl21. (Ccl21b); PD-L1 and Mfge8; PD-L1 and Serpin B9 (Spi6); HLA-G (H2-M3) and Cd47; HLA-G (H2-M3) and Cd200; HLA-G (H2-M3). And FASLG (FasL); HLA-G (H2-M3) and Ccl21 (Ccl21b); HLA-G (H2-M3) and Mfge8; HLA-G (H2-M3) and Serpin B9 (Spi6); Cd47 and Cd200; Cd47 and FASLG (FasL); Cd47 and Ccl21 (Ccl21b); Cd47 and Mfge8; Cd47 and Serpin B9 (Spi6); Cd200 and FASLG (FasL); Cd200 and Ccl21 (Ccl21b); Cd200 and Mdge and Cd200 and Mfge8Cs. ); FASLG(FasL) and Ccl21(Ccl21b); FASLG(FasL) and Mfge8; FASLG(FasL) and Serpin B9(Spi6); Ccl21(Ccl21b) and Mfge8; And Serpin B9 (Spi6)).

In some embodiments of any of the aforementioned aspects, the cells are transgene PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9. Three of the (Spi6) sets (eg PD-L1, HLA-G (H2-M3) and Cd47; PD-L1, HLA-G (H2-M3) and Cd200; PD-L1, HLA-G (H2. -M3) and FASLG(FasL); PD-L1, HLA-G(H2-M3) and Ccl21(Ccl21b); PD-L1, HLA-G(H2-M3) and Mfge8; PD-L1, HLA-G( H2-M3) and Serpin B9 (Spi6); PD-L1, Cd47 and Cd200; PD-L1, Cd47 and FASLG (FasL); PD-L1, Cd47 and Ccl21(Ccl21b); PD-L1, Cd47 and Mfge8; PD. -L1, Cd47 and Serpin B9; PD-L1, Cd200 and FASLG (FasL); PD-L1, Cd200 and Ccl21 (Ccl21b); PD-L1, Cd200 and Mfge8; PD-L1, Cd200 and Serpin B9 (Spi6); PD-L1, FASLG(FasL) and Ccl21(Ccl21b); PD-L1, FASLG(FasL) and Mfge8; PD-L1, FASLG(FasL) and Serpin B9(Spi6); PD-L1, Ccl21(Ccl21b) and Mcl21(Ccl21b). PD-L1, Ccl21 (Ccl21b) and Serpin B9 (Spi6); PD-L1, Mfge8 and Serpin B9 (Spi6); HLA-G (H2-M3), Cd47 and Cd200; HLA-G (H2-M3), Cd47 and FASLG (FasL); HLA-G (H2-M3), Cd47 and Ccl21 (Ccl21b); HLA-G (H2-M3), Cd47 and Mfge8; HLA-G (H2-M3), Cd47 and Serpin B9; HLA-G (H2-M3), Cd200 and FASLG (FasL); HLA-G (H2-M3), Cd200 and Ccl21 (Ccl21b); HLA-G (H2-M3), Cd200 and Mfge8; HLA-G (H2). -M3), Cd200 and Serpin B9; HLA-G (H2-M3), FASLG (FasL) and Ccl. 21(Ccl21b); HLA-G(H2-M3), FASLG(FasL) and Mfge8; HLA-G(H2-M3), FASLG(FasL) and Serpin B9(Spi6); HLA-G(H2-M3), Ccl21 (Ccl21b) and Mfge8; HLA-G (H2-M3), Ccl21 (Ccl21b) and Serpin B9 (Spi6); HLA-G (H2-M3), Mfge8 and Serpin B9 (Spi6); Cd47, Cd200 and FAd. FasL); Cd47, Cd200 and Ccl21 (Ccl21b); Cd47, Cd200 and Mfge8; Cd47, Cd200 and Serpin B9 (Spi6); Cd47, FASLG (FasL) and Ccl21 (Ccl21b); , FASLG (FasL) and Serpin B9 (Spi6); Cd47, Ccl21 (Ccl21b) and Mfge8; Cd47, Ccl21 (Ccl21b) and Serpin B9 (Spi6); Cd47, Mfge8 and Serpin B; Serpin B9 (Spi6); And Ccl21 (Ccl21b); Cd200, FASLG (FasL) and Mfge8; Cd200, FASLG (FasL) and Serpin B9 (Spi6); Cd200, Ccl21 (Ccl21b) and Mfge8; Cd200, Mfge8 and Serpin B9 (Spi6); FASLG (FasL), Ccl21 (Ccl21b) and Mfge8; FASLG (FasL), Ccl21 (Ccl21b) and Serpin B9 (Spi6); )including.

In some embodiments of any of the aforementioned aspects, the cells are transgene PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9. Four of the (Spi6) sets (eg PD-L1, HLA-G (H2-M3), Cd47 and Cd200; PD-L1, HLA-G (H2-M3), Cd47 and FASLG (FasL); PD- L1, HLA-G (H2-M3), Cd47 and Ccl21 (Ccl21b); PD-L1, HLA-G (H2-M3), Cd47 and Mfge8; PD-L1, HLA-G (H2-M3), Cd47 and Serpin B9 (Spi6); PD-L1, HLA-G (H2-M3), Cd200 and FASLG (FasL); PD-L1, HLA-G (H2-M3), Cd200 and Ccl21 (Ccl21b); PD-L1, HLA-G (H2-M3), Cd200 and Mfge8; PD-L1, HLA-G (H2-M3), Cd200 and Serpin B9 (Spi6); PD-L1, HLA-G (H2-M3), FASLG (FasL). ) And Ccl21 (Ccl21b); PD-L1, HLA-G (H2-M3), FASLG (FasL) and Mfge8; PD-L1, HLA-G (H2-M3), FASLG (FasL) and Serpin B9 (Spi6). PD-L1, HLA-G (H2-M3), Ccl21 (Ccl21b) and Mfge8; PD-L1, HLA-G (H2-M3), Ccl21 (Ccl21b) and Serpin B9 (Spi6); PD-L1, HLA -G(H2-M3), Mfge8 and Serpin B9(Spi6); PD-L1, Cd47, Cd200 and FASLG(FasL); PD-L1, Cd47, Cd200 and Ccl21(Ccl21b); PD-L1, Cd47, Cd200 and. Mfge8; PD-L1, Cd47, Cd200 and Serpin B9 (Spi6); PD-L1, Cd47, FASLG (FasL) and Ccl21 (Ccl21b); PD-L1, Cd47, FASLG (FasL) and Mfge8; PD-L1, Cd. , FASLG (FasL) and Serpin B9 (Spi6); PD-L1, Cd47, Ccl21 (Ccl21b) and Mfge8; PD-L1, Cd47, Cc. 121 (Ccl21b) and Serpin B9 (Spi6); PD-L1, Cd47, Mfge8 and Serpin B9 (Spi6); PD-L1, Cd200, FASLG (FasL) and Ccl21 (Ccl21b); PD-L1, Cd200, FASLG, FASGL, FASL. ) And Mfge8; PD-L1, Cd200, FASLG (FasL) and Serpin B9 (Spi6); PD-L1, Cd200, Ccl21 (Ccl21b) and Mfge8; PD-L1, Cd200, Ccl21 (Ccl21b) and Serpin B9 (Serpin 6). PD-L1, Cd200, Mfge8 and Serpin B9 (Spi6); PD-L1, FASLG (FasL), Ccl21 (Ccl21b) and Mfge8; PD-L1, FASLG (FasL), Ccl21 (Ccl21b) and Serpin. PD-L1, FASLG (FasL), Mfge8 and Serpin B9 (Spi6); PD-L1, Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6); HLA-G (H2-M3), Cd47, Cd200 and FASLG (Fd). FasL); HLA-G (H2-M3), Cd47, Cd200 and Ccl21 (Ccl21b); HLA-G (H2-M3), Cd47, Cd200 and Mfge8; HLA-G (H2-M3), Cd47, Cd200 and Serpin. B9 (Spi6); HLA-G (H2-M3), Cd47, FASLG (FasL) and Ccl21 (Ccl21b); HLA-G (H2-M3), Cd47, FASLG (FasL) and Mfge8; HLA-G (H2-). M3), Cd47, FASLG (FasL) and Serpin B9 (Spi6); HLA-G (H2-M3), Cd47, Ccl21 (Ccl21b) and Mfge8; HLA-G (H2-M3), Cd47, Ccl21 (Ccl21b) and. Serpin B9 (Spi6); HLA-G (H2-M3), Cd47, Mfge8 and Serpin B9 (Spi6); HLA-G (H2-M3), Cd200, FASLG (FasL) and Ccl21 (Ccl21b); HLA-G (HLA-G). H2-M3), Cd200, FASLG(FasL) and Mfge8; HLA-G(H2-M3), Cd200, FASLG(FasL) and Serpin B. 9(Spi6); HLA-G(H2-M3), Cd200, Ccl21(Ccl21b) and Mfge8; HLA-G(H2-M3), Cd200, Ccl21(Ccl21b) and Serpin B9(Spi6); HLA-G(H2). -M3), Cd200, Mfge8 and Serpin B9(Spi6); HLA-G(H2-M3), FASLG(FasL), Ccl21(Ccl21b) and Mfge8; HLA-G(H2-M3), FASLG(FasL), Ccl21. (Ccl21b) and Serpin B9(Spi6); HLA-G(H2-M3), FASLG(FasL), Mfge8 and Serpin B9(Spi6); HLA-G(H2-M3), Ccl21(Ccl21b), Mfge8 and Ser. (Spi6); Cd47, Cd200, FASLG(FasL) and Ccl21(Ccl21b); Cd47, Cd200, FASLG(FasL) and Mfge8; Cd47, Cd200, FASLG(FasL) and Serpin B9(Spi6d; Ccl21b) and Mfge8; Cd47, Cd200, Ccl21(Ccl21b) and Serpin B9 (Spi6); Cd47, Cd200, Mfge8 and Serpin B9 (Spi6); FasL), Ccl21(Ccl21b) and Serpin B9(Spi6); Cd47, Ccl21(Ccl21b), Mfge8 and Serpin B9(Spi6); Ccl21 (Ccl21b) and Serpin B9 (Spi6); Cd200, Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6); or FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin.

In some embodiments of any of the above aspects, the cells are transgene PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9. Five of the (Spi6) sets (eg PD-L1, HLA-G (H2-M3), Cd47, Cd200 and FASLG (FasL); PD-L1, HLA-G (H2-M3), Cd47, Cd200 and Ccl21 (Ccl21b); PD-L1, HLA-G (H2-M3), Cd47, Cd200 and Mfge8; PD-L1, HLA-G (H2-M3), Cd47, Cd200 and Serpin B9 (Spi6); PD-L1 , HLA-G(H2-M3), Cd47, FASLG(FasL) and Ccl21(Ccl21b); PD-L1, HLA-G(H2-M3), Cd47, FASLG(FasL) and Mfge8; PD-L1, HLA-. G(H2-M3), Cd47, FASLG(FasL) and Serpin B9(Spi6); PD-L1, HLA-G(H2-M3), Cd47, Ccl21(Ccl21b) and Mfge8; PD-L1, HLA-G( H2-M3), Cd47, Ccl21 (Ccl21b) and Serpin B9 (Spi6); PD-L1, HLA-G (H2-M3), Cd47, Mfge8 and Serpin B9 (Spi6); PD-L1, HLA-G (H2). -M3), Cd200, FASLG(FasL) and Ccl21(Ccl21b); PD-L1, HLA-G(H2-M3), Cd200, FASLG(FasL) and Mfge8, PD-L1, HLA-G(H2-M3). , Cd200, FASLG(FasL) and Serpin B9(Spi6); PD-L1, HLA-G(H2-M3), Cd200, Ccl21(Ccl21b) and Mfge8; PD-L1, HLA-G(H2-M3), Cd200. , Ccl21 (Ccl21b) and Serpin B9 (Spi6); PD-L1, HLA-G (H2-M3), Cd200, Mfge8 and Serpin B9 (Spi6); PD-L1, HLA-G (H2-M3), FASLG ( FasL), Ccl21 (Ccl21b) and Mfge8; PD-L1, HLA-G (H2-M3), FASLG (FasL), Ccl21 (Ccl21b) and Serp. in B9(Spi6); PD-L1, HLA-G(H2-M3), FASLG(FasL), Mfge8 and Serpin B9(Spi6); PD-L1, HLA-G(H2-M3), Ccl21(Ccl21b), Mfge8 and Serpin B9 (Spi6); PD-L1, Cd47, Cd200, FASLG (FasL) and Ccl21 (Ccl21b); PD-L1, Cd47, Cd200, FASLG (FasL) and Mfge8, PD-L1, Cd47, Cd47, Cd47, Cd47, Gd200. (FasL) and Serpin B9 (Spi6); PD-L1, Cd47, Cd200, Ccl21 (Ccl21b) and Mfge8; PD-L1, Cd47, Cd200, Ccl21 (Ccl21b) and Serpin B9 (Spi6); PD, L1, C1. Cd200, Mfge8 and Serpin B9 (Spi6); PD-L1, Cd47, FASLG (FasL), Ccl21 (Ccl21b) and Mfge8; PD-L1, Cd47, FASLG (FasL), Ccl21 (Ccl21b) and Serpin. PD-L1, Cd47, FASLG(FasL), Mfge8 and Serpin B9(Spi6); PD-L1, Cd47, Ccl21(Ccl21b), Mfge8 and Serpin B9(Spi6); PD-L1, Cd200, FASLG, FasLG(FasL), FasL. (Ccl21b) and Mfge8; PD-L1, Cd200, FASLG(FasL), Ccl21(Ccl21b) and Serpin B9(Spi6); PD-L1, Cd200, FASLG(FasL), Mfge8 and Serpin B9(Spi1); , Cd200, Ccl21(Ccl21b), Mfge8 and Serpin B9(Spi6); PD-L1, FASLG(FasL), Ccl21(Ccl21b), Mfge8 and Serpin B9(Spi6); HLA-G(Hd2-C3). , FASLG(FasL) and Ccl21(Ccl21b); HLA-G(H2-M3), Cd47, Cd200, FASLG(FasL) and Mfge8; HLA-G(H2-M3), Cd47, Cd200, FASLG(FasL) and Serpin. B9 (Spi6); HLA-G (H2-M3), Cd47, Cd20. 0, Ccl21 (Ccl21b) and Mfge8; HLA-G (H2-M3), Cd47, Cd200, Ccl21 (Ccl21b) and Serpin B9 (Spi6); HLA-G (H2-M3), Cd47, Cd200, MfgeB9 and Serpine. (Spi6); HLA-G(H2-M3), Cd47, FASLG(FasL), Ccl21(Ccl21b) and Mfge8; HLA-G(H2-M3), Cd47, FASLG(FasL), Ccl21(Ccl21b) and Serpin B9. (Spi6); HLA-G (H2-M3), Cd47, FASLG (FasL), Mfge8 and Serpin B9 (Spi6); HLA-G (H2-M3), Cd47, Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6). ); HLA-G(H2-M3), Cd200, FASLG(FasL), Ccl21(Ccl21b) and Mfge8; HLA-G(H2-M3), Cd200, FASLG(FasL), Ccl21(Ccl21b) and Serpin B9(Spi6). ); HLA-G (H2-M3), Cd200, FASLG (FasL), Mfge8 and Serpin B9 (Spi6); HLA-G (H2-M3), Cd200, Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6); HLA-G(H2-M3), FASLG(FasL), Ccl21(Ccl21b), Mfge8 and Serpin B9(Spi6); Cd47, Cd200, FASLG(FasL), Ccl21(Ccl21b) and Mfge8; Cd47, Cd47, Cd47, Cd47, Cd47, Cd47, Cd47, Cd200. ), Ccl21 (Ccl21b) and Serpin B9 (Spi6); Cd47, Cd200, FASLG (FasL), Mfge8 and Serpin B9 (Spi6); (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6); or Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6). In some embodiments of any of the above aspects, the cells are transgene PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9. Six of the (Spi6) sets (eg PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL) and Ccl21 (Ccl21b); PD-L1, HLA-G (H2-M3)). , Cd47, Cd200, FASLG(FasL) and Mfge8; PD-L1, HLA-G(H2-M3), Cd47, Cd200, FASLG(FasL) and Serpin B9(Spi6); PD-L1, HLA-G(H2-). M3), Cd47, Cd200, Ccl21(Ccl21b) and Mfge8; PD-L1, HLA-G(H2-M3), Cd47, Cd200, Ccl21(Ccl21b) and Serpin B9(Spi6); PD-L1, HLA-G( H2-M3), Cd47, Cd200, Mfge8 and Serpin B9 (Spi6); PD-L1, HLA-G (H2-M3), Cd47, FASLG (FasL), Ccl21 (Ccl21b) and Mfge8; PD-L1, HLA-. G(H2-M3), Cd47, FASLG(FasL), Ccl21(Ccl21b) and Serpin B9(Spi6); PD-L1, HLA-G(H2-M3), Cd47, FASLG(FasL), Mfge8 and Serpin B9(Ser). Spi6); PD-L1, HLA-G (H2-M3), Cd200, FASLG (FasL), Ccl21 (Ccl21b) and Mfge8; PD-L1, HLA-G (H2-M3), Cd200, FASLG (FasL), Ccl21 (Ccl21b) and Serpin B9 (Spi6); PD-L1, HLA-G (H2-M3), Cd200, FASLG (FasL), Mfge8 and Serpin B9 (Spi6); PD-L1, HLA-G (H2-M3). ), Cd200, Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6); PD-L1, Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b) and Mfge8; PD-L1, Cd47, Cd200, FASLG (FASLG). Ccl21 (Ccl21b) and Serpin B9 (S pi6); PD-L1, Cd47, Cd200, FASLG (FasL), Mfge8 and Serpin B9 (Spi6); PD-L1, Cd47, Cd200, Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6)d-PD-L; , FASLG(FasL), Ccl21(Ccl21b), Mfge8 and Serpin B9(Spi6); PD-L1, Cd200, FASLG(FasL), Ccl21(Ccl21b), Mfge8 and Serpin B9(Spi6)(HPI6); ), Cd47, Cd200, FASLG(FasL), Ccl21(Ccl21b) and Mfge8; HLA-G(H2-M3), Cd47, Cd200, FASLG(FasL), Ccl21(Ccl21b) and Serpin B9(Spi6); (H2-M3), Cd47, Cd200, FASLG(FasL), Mfge8 and Serpin B9(Spi6); HLA-G(H2-M3), Cd47, Cd200, Ccl21(Ccl21b), Mfge8 and Serpin B9(Spi6); -G(H2-M3), Cd47, FASLG(FasL), Ccl21(Ccl21b), Mfge8 and Serpin B9(Spi6); HLA-G(H2-M3), Cd200, FASLG(FasL), Ccl21(Ccl21b), Mfge. And Serpin B9 (Spi6); or Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6)).

In some embodiments of any of the aforementioned aspects, the cells are transgene PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9. 7 of the (Spi6) set (eg PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b) and Mfge8; PD-L1, HLA-G (H2-). M3), Cd47, Cd200, FASLG(FasL), Ccl21(Ccl21b) and Serpin B9(Spi6); PD-L1, HLA-G(H2-M3), Cd47, Cd200, FASLG(FasL), Mfge8 and Serpin B(Serpin B). Spi6); PD-L1, HLA-G (H2-M3), Cd47, Cd200, Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6); PD-L1, HLA-G (H2-M3), Cd47, FASLG (Spi6). FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6); PD-L1, HLA-G (H2-M3), Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Serpin B9). -L1, Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6); or HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Ccl21 (Ccl21b). And Serpin B9 (Spi6)).

In some embodiments of any of the above aspects, the cells are transgene PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9. Includes all eight of the (Spi6) set.

In some embodiments of any of the foregoing aspects, the cells are of transgene HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6). It includes one or more of the sets (eg, one, two, three, four, five, six or all seven).

In some embodiments of any of the foregoing aspects, the cells are set of transgene PD-L1, HLA-G (H2-M3), Cd47, Cd200, Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6). One or more of (eg, 1, 2, 3, 4, 5, 6, or all 7).

In some embodiments of any of the above aspects, the cells are one of the set of transgene HLA-G (H2-M3), Cd47, Cd200, Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6). Multiple (eg, one, two, three, four, five or all six) are included.

In some embodiments of any of the aforementioned aspects, the cells have not been modified to express PD-L1.

In some embodiments of any of the above aspects, the cells are not modified to express FasL.

In some embodiments of any of the above aspects, the cells are not modified to express TGF-β. In some embodiments of any of the above aspects, the cells are not modified to express CTLA4 or CLTA4-Ig. In some embodiments of any of the above aspects, the cells have not been modified to express IDO. In some embodiments of any of the above aspects, the cells have not been modified to express IL-35. In some embodiments of any of the above aspects, the cells have not been modified to express IL-10. In some embodiments of any of the above aspects, the cells have not been modified to express VEGF. In some embodiments of any of the above aspects, the cells are not modified to express NFκb decoy receptor F. In some embodiments of any of the above aspects, the cells are not modified to express soluble TNFR. In some embodiments of any of the above aspects, the cells are not modified to express CCR7. In some embodiments of any of the above aspects, the cells are not modified to express SOCS1. In some embodiments of any of the above aspects, the cells are not modified to express HLA-E. In some embodiments of any of the above aspects, the cells are not modified to express a siRNA directed against IL-12.

Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

As used herein, the term "about" refers to a value that is 10% above or below the stated value. For example, the term "about 5 nM" indicates a range of 4.5 nM to 5.5 nM.

As used herein, the term "activated leukocytes" refers to leukocytes (e.g., granulocytes such as neutrophils, eosinophils or basophils, monocytes or Lymphocytes such as B or T cells). When leukocytes are activated, they proliferate, secrete cytokines, differentiate, present antigens, become more polarized, more phagocytic, and/or more cytotoxic. Factors that stimulate the activation of immune cells include proinflammatory cytokines, pathogens and non-self antigen presentation. Activated leukocytes can be isolated from lymphoid organs. Leukocytes such as T cells can also be activated in vitro using anti-CD3/CD28 beads or other methods used by those of skill in the art (eg, Frauwith and Thompson, J. Clin Invest 109:295-299 (2002). And Trickett and Kwan, J Immunol Methods 275:251-255 (2003)).

As used herein, "homogeneous" means cells, tissues, DNA or factors taken from or derived from different subjects of the same species.

As used herein, the term "stem cell" refers to a cell that is capable of differentiating into one or more specialized cells and has the capacity for self-renewal. Stem cells include pluripotent stem cells (PSCs) such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), and pluripotent cells such as cord blood stem cells, mesenchymal stromal cells and adult stem cells found in various tissues. Includes sex stem cells. The term "stem cell" refers to cells that are subject to genome editing, cells that can serve as a source of therapeutic cell types (eg, lineage-restricted cells or terminally differentiated cells used in cell therapy or cells of the desired target tissue) and " Also included are cells that have “artificial” cell-acquiring stem cell properties (eg, pluripotent or pluripotent or self-renewing).

As used herein, the terms "embryonic stem cell" and "ES cell" refer to an embryonic-derived totipotent stem cell or multipotent stem cell derived from an inner cell mass of a blastocyst that can be maintained in in vitro culture under suitable conditions. Refers to competent stem cells. ES cells can differentiate into cells of any of the three vertebrate germ layers, eg, endoderm, ectoderm or mesoderm. ES cells are also characterized by their ability to grow indefinitely under suitable in vitro culture conditions. For example, Thomson et al. , Science 282:1145 (1998).

As used herein, the terms “induced pluripotent stem cells”, “iPS cells” and “iPSCs” refer to pluripotent stem cells that can be directly derived from differentiated somatic cells. For example, Oct3/4, Sox family transcription factor (for example, Sox1, Sox2, Sox3, Soxl5), Myc family transcription factor (for example, c-Myc, 1-Myc, n-Myc), Kruppel-like family (KLF) transcription factor. (Eg, KLF1, KLF2, KLF4, KLF5) and/or related transcription factors, such as NANOG, LIN28 and/or Glis1, by introducing into the non-pluripotent cell a particular set of reprogramming factors. iPS cells can be generated. Human iPS cells can also be generated, for example, by using miRNA, a small molecule that mimics the action of transcription factors, or lineage determinants. Human iPS cells are characterized by the ability to differentiate into any of the three vertebrate germ layers, eg endoderm, ectoderm or mesoderm. Human iPS cells are also characterized by their ability to grow indefinitely under suitable in vitro culture conditions. See, for example, Takahashi and Yamanaka, Cell 126:663 (2006).

As used herein, the term "relax activation and function of an antigen presenting cell" is a gene product, the function of which promotes activation of an antigen presenting cell or activation of a leukocyte attack graft. It refers to a transgene that encodes a gene product that inhibits the ability of the antigen-presenting cells to react (Fiorentino et al., J Immunol. 146:3444-51 (1991); Sario et al., Eur J Immunol. 29). : 3245-53 (1999)). In embodiments, relaxation of antigen presenting cell activation and function is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% of APC activation and function relative to a control. Refers to a 90%, 95% or 100% reduction (eg as determined using an assay for antigen presenting cell activation, eg reduced proliferation, reduced pro-inflammatory cytokines (eg interleukin-1 (IL- 1, eg IL-1β), IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-18, tumor necrosis factor (TNF, eg TNFα), interferon γ (IFNγ). ) And secretion of granulocyte-macrophage colony stimulating factor (GMCSF), which can be measured using ELISA or Western blot analysis of patient samples such as culture medium or blood) or cell surface markers (eg CD11c, Decreased levels of CD11b, HLA molecules (eg MHC-II), CD40, B7, IL-2, CD80 or CD86 (which include flow cytometry, immunohistochemistry, in situ hybridization and measurement of cell surface markers). Can be evaluated using other assays that allow), etc.) Antigen presenting cells include dendritic cells, B cells and macrophages. Mast cells and neutrophils are also induced to present antigen. Methods for determining activation and function relaxation of antigen presenting cells are known in the art.Examples of gene products that relax activation and function of antigen presenting cells include Ccl21 (Ccl21b) and PD-. Such transgenes may be referred to herein as “cloaking” or “cloaked” genes, including but not limited to L1.

As used herein, the term "relax activity or cytolytic function of graft-attacking leukocytes" is a gene product whose function is the activity of graft-attacking leukocytes in the vicinity of allograft cells. Alternatively, it refers to a transgene encoding a gene product that is to inhibit or prevent the cytolytic function (MacDonald et al., J Immunol. 126:1671-5 (1981); Bongrand et al., Eur J Immunol. 13). : 424-9 (1983); MacDonald et al., Eur J Immunol. 9:466-70 (1979)). In one embodiment, activation of the graft-attacking leukocytes or relaxation of the cytolytic function is at least about 10%, 20%, 30%, 40%, 50%, 60% of the activation or cytolytic function of the leukocytes relative to the control. , 70%, 80%, 90%, 95% or 100% reduction (eg, decreased proliferation, decreased pro-inflammatory cytokines (eg, interleukin) as measured using an assay for leukocyte activation. -1 (IL-1 such as IL-1β), IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-18, tumor necrosis factor (TNF such as TNFα). , Secretion of interferon-γ (IFNγ) and granulocyte-macrophage colony-stimulating factor (GMCSF), which can be measured using ELISA or Western blot analysis of patient samples such as culture medium or blood) or reduced polarization (For example, decreased levels of IL-12, TNF, IL-1β, IL-6, IL-23, MARCO, MHC-II, CD86, iNOS, CXCL9 and CXCL10 in macrophages or monocytes or Th1 specific markers (eg. , T-bet, IL-12R, STAT4), chemokine receptors (eg CCR5, CXCR6 or CXCR3); or Th2-specific markers in T cells (eg CCR3, CXCR4, STAT6, GATA3 or IL-4Rα). Decreased levels, which are assessed using flow cytometry, immunohistochemistry, in situ hybridization, qPCR or Western blot analysis of "cell surface markers or intracellular proteins" and secreted protein ELISA or Western blot analysis Can be used) or using an assay for cytolytic function (eg, incubating leukocytes with a target cell line pre-coated with an antibody against a surface antibody expressed by the target cell line to detect viable target cells with fluorescent viability staining). Determined by measuring the number or by measuring the secretion of cytolytic granules from leukocytes (eg perforin, granzymes or other cytolytic proteins released by immune cells)). Methods for determining activation of transplant-challenged leukocytes or relaxation of cytolytic function are known in the art. Examples of gene products that alleviate the activation or cytolytic function of transplant-challenged leukocytes include, but are not limited to, PD-L1, HLA-G(H2-M3), Cd39, Cd73 and Lag3. Such transgenes may be referred to herein as "cloaking" or "cloaked" genes.

As used herein, the term "alleviating macrophage cytolytic function and phagocytosis of allograft cells" is a gene product whose function is the macrophage cytolytic function of allograft cells and/or Refers to a transgene that encodes a gene product that is to inhibit or prevent phagocytosis (Fish et al., Toxicology. 19:127-38. (1981); Sung et al., J Biol Chem. 260:546. -54 (1985); Amash et al., J Immunol. 196:3331-40 (2016)). In embodiments, alleviating macrophage cytolytic function and phagocytosis of allograft cells is at least about 10%, 20%, 30%, 40% of macrophage cytolytic function and/or phagocytosis of allograft cells relative to a control. , 50%, 60%, 70%, 80%, 90%, 95% or 100% reduction (eg as determined using an assay for macrophage cytolytic function (eg expressed by the target cell line) By incubating macrophages with a target cell line pre-coated with an antibody against a surface antigen and measuring the number of viable target cells with fluorescent viability staining, or by cytolytic (sex) granules released from macrophages (eg perforin , Granzymes or other cytolytic (sex) proteins released by immune cells, or determined using assays for macrophage phagocytosis (eg expressed by fluorescent beads or target cell lines). Macrophages are cultured with a target cell line pre-coated with antibodies against surface antigens to measure the fluorescence inside immune cells or to quantify the number of beads or phagocytosed cells)). Methods for determining relaxation of macrophage cytolytic function and phagocytosis are known in the art.Examples of gene products that attenuate macrophage cytolytic function include Cd47, Cd200, Mfge8 and Il1r2. Such transgenes may be referred to herein as "cloaking" or "cloaked" genes.

As used herein, the term “induce apoptosis in graft-attacking leukocytes” is a gene product whose function is to kill graft-attacking leukocytes in the vicinity of the graft cells. Refers to a transgene encoding a gene product (Huang et al., Proc Natl Acad Sci US A. 96:14871-6 (1999); Suzuki et al., Proc Natl Acad Sci U S A. 97:1707-12). (2000); Simon et al., Proc Natl Acad Sci US A. 98:5158-63 (2001)). In embodiments, the induction of apoptosis in graft-attacked leukocytes is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% of the induction of apoptosis in graft-attacked leukocytes relative to controls. , 90%, 95% or 100% increase (as determined using assays for apoptosis, such as the use of TUNEL staining, caspase staining or Annexin V staining or fluorescent viability staining). Methods for determining the induction of apoptosis in graft-challenged leukocytes are known in the art. Examples of gene products capable of inducing apoptosis in graft-attacked leukocytes include, but are not limited to, FASLG (FasL) and Tnfsf10. Such transgenes may be referred to herein as "cloaking" or "cloaked" genes.

As used herein, the term "relaxing a local immune protein" is a gene product, the function of which is to inhibit the activity of a local protein, the function of which is the graft attack. Refers to a transgene encoding a gene product that promotes leukocyte accumulation and/or their cytolytic function (Felix et al., Nat Rev Immunol. 17:112-29 (2017)). In embodiments, relief of local immune protein is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% of local inflammatory protein relative to controls. Or 100% reduction (eg, inflammatory proteins that promote leukocyte activation or migration to sites of inflammation (eg, chemokines such as CCL2, CCL3, CCL5, CXCL1, CXCL2 and CXCL8 or proinflammatory cytokines such as IL). -1β, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-18, TNFα, IFNγ or GMCSF, which can be assayed by ELISA, Western blot analysis or secreted proteins. Can be measured using other techniques known in the art) as determined). Methods for determining the relief of local inflammatory proteins are known in the art. Examples of gene products that mitigate local inflammatory proteins include, but are not limited to, PD-L1, Il1r2 and Ackr2. Such transgenes may be referred to herein as "cloaking" or "cloaked" genes.

As used herein, the term "protects against leukocyte-mediated apoptosis" is a gene product whose function is to inhibit any cellular component that can induce apoptosis or cytolysis of allograft cells. Is referred to (Abdullah et al., J Immunol. 178:3390-9 (2007)). In embodiments, protection against leukocyte-mediated apoptosis is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100 of leukocyte-mediated apoptosis relative to a control. % Reduction (eg, as determined using an assay for leukocyte-mediated apoptosis (eg, incubating leukocytes with a target cell line pre-coated with an antibody to the surface antigen expressed by the target cell line, fluorescent viability staining). By measuring the number of viable target cells having, or the number of cytolytic (sex) granules released from leukocytes (eg, perforin, granzymes or other cytolytic (sex) proteins released from immune cells). Methods for determining protection against leukocyte-mediated apoptosis are known in the art. Examples of gene products that protect against leukocyte-mediated apoptosis include Serpin B9 (Spi6) and Dad1. Such a transgene may be referred to herein as a "cloaking" or "cloaked" gene.

As used herein, the term "biological material" refers to designed polypeptides and the corresponding coding DNA that can be expressed as a transgene. The polypeptide is capable of activating or inhibiting the function of an endogenous gene or inhibiting or activating a biological process. Methods for determining whether a polypeptide has agonist or antagonist activity or function are generally known in the art. In embodiments, the agonist function is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90, 95% or 100% of the function relative to the control function. is there. In embodiments, the antagonist function is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90, 95% or 100% of the function relative to the control function. is there.

As used herein, the term "operably linked" refers to a first molecule linked to a second molecule, the first molecule affecting the function or expression of the second molecule. The molecules are arranged to give. The two molecules may or may not be part of a single continuous molecule and may or may not be adjacent. For example, a promoter is operably linked to a transcribable polynucleotide molecule if the promoter regulates transcription of the transcribable polynucleotide molecule of interest in the cell. Furthermore, two parts of a transcriptional regulatory element are operably linked to each other when the transcriptional activation function of one part is linked so that it is not adversely affected by the presence of the other part. The two transcriptional regulatory elements can be operably linked to each other by a linker nucleic acid (eg, an intervening non-coding nucleic acid) or can be operably linked to each other without the presence of intervening nucleotides.

As used herein, the term “promoter” refers to a recognition site on DNA that is bound by RNA polymerase. The polymerase drives transcription of the allograft.

"Percent (%) sequence identity" in reference to a reference polynucleotide or polypeptide sequence refers to the reference polynucleotide after aligning the sequences as necessary to introduce gaps to achieve maximum percent sequence identity. It is defined as the proportion of the nucleic acid or amino acid in the candidate sequence that is identical to the nucleic acid or amino acid in the nucleotide or polypeptide sequence. Alignments for the purpose of determining percent nucleic acid or amino acid sequence identity use a variety of methods within the ability of those skilled in the art, for example, publicly available computer software such as BLAST, BLAST-2 or Megalign software. Can be achieved. One of ordinary skill in the art can determine the appropriate parameters, including any algorithm for aligning sequences, necessary to achieve maximal alignment over the entire length of the sequences being compared. For example, percent sequence identity values can be generated using the sequence comparison computer program BLAST. By way of example, the percent sequence identity of a given nucleic acid or amino acid sequence A to a given nucleic acid or amino acid sequence B, with or to B (which, in turn, to a given nucleic acid or amino acid sequence B, A given nucleic acid or amino acid sequence A with a certain percent sequence identity to or with B) can be expressed as A) is calculated as follows.
100 x (ratio X/Y)
Where X is the number of nucleotides or amino acids scored by the sequence alignment program (eg, BLAST) as the same match in the A and B alignments of the program, and Y is the total number of nucleic acids in B. It is understood that when the length of the nucleic acid or amino acid sequence A is not equal to the nucleic acid or amino acid sequence B, the percent sequence identity of A to B is not equal to the percent sequence identity of B to A.

As used herein, the term "pharmaceutical composition" refers to a subject, e.g., a mammal, e.g., a human, to prevent, treat or control a particular disease or condition that the subject has or may develop. Refers to a mixture comprising a therapeutic agent to be administered, optionally in combination with one or more pharmaceutically acceptable excipients, diluents and/or carriers.

As used herein, the term "pharmaceutically acceptable" does not have excessive toxicity, irritation, allergic reactions and/or complications of other problems commensurate with the ideal benefit/risk ratio. , Refers to a compound, material, composition and/or dosage form suitable for contact with a subject, eg, mammalian (eg, human) tissue.

As used herein, the term "wild type" refers to the genotype that has the highest frequency for a particular gene in a given organism.

As used herein, the terms “one cell division locus”, “plurality of cell division loci” and “CDL” are genomic loci, the transcripts of which are expressed by dividing cells. Refers to a locus. When the CDL comprises a single locus, the absence of CDL expression in the cell (or derivative thereof) is ablated in the absence of CDL expression, or proliferation of the cell in the absence of CDL expression. Tumor initiation and/or formation is impeded as it is either blocked or impaired. When the CDL comprises multiple loci, the absence of expression by all or a subset of the loci in the cell (or derivative thereof) is either ablated in the absence of CDL expression, or the proliferation of the cells is CDL. It is either blocked or impaired in the absence of expression, thus impeding tumor initiation and/or formation. CDL may or may not be expressed in non-dividing and/or non-proliferating cells. The CDL can be endogenous to the host cell or transgene. If the CDL is a transgene, it can be from the same or a different species as the host cell, or it can be of synthetic origin. In one embodiment, the CDL is a single locus that is transcribed during cell division. For example, in one embodiment, the single locus CDL is CDK1. In one embodiment, the CDL comprises more than one locus transcribed during cell division. For example, in one embodiment, the multilocus CDL comprises two MYC genes (c-Myc and N-myc) (Scognamiglio et al., 2016). In one embodiment, the multilocus CDL comprises AURORA B and C kinases, which may have overlapping functions (Fernandez-Miranda et al., 2011). Cell division and cell proliferation are terms that can be used interchangeably herein.

As used herein, the terms “normal rate of cell division”, “normal cell division rate”, “normal rate of cell proliferation” and “normal cell proliferation rate” are typical of non-cancerous healthy cells. Refers to the rate of cell division and/or proliferation. The normal rate of cell division and/or proliferation can be cell type specific. For example, it is widely accepted that cell numbers in the epidermis, intestine, lung, blood, bone marrow, thymus, testis, uterus and mammary gland are maintained by fast cell division and fast cell death. In contrast, cell numbers in the pancreas, kidney, cornea, prostate, bone, heart and brain are maintained by slow cell division and slow cell death (Pellettieri and Sanchez Alvarado, 2007).

As used herein, the terms "inducible negative proliferative effector" and "iNEP" are used to either i) stop or block CDL expression by induction, thereby preventing cell division and proliferation, or ii) by inducing CDL-expressing cells. Rate of cell division relative to normal cell division rate such that the rate of cell division that ablates (ie kills at least a portion of the proliferating cells) or iii) is not fast enough to contribute to tumorigenesis Slowing down refers to genetic modifications that facilitate the use of CDL expression for the purpose of controlling cell division and/or proliferation.

As used herein, the terms "ablation link" and "ALINK" refer to an example of iNEP that contains a transcriptional link between the CDL and a sequence encoding a negative selection marker. ALINK modification allows a user to inducibly kill proliferating host cells containing ALINK or kill at least a portion of the proliferating cells by exposing the ALINK-modified cells to an inducer of a negative selection marker, It becomes possible to inhibit the growth of host cells. For example, cells that have been modified to contain ALINK in CDL to induce ablative cells by killing at least a portion of the proliferating cells or to inhibit cell proliferation are treated with an inducer of a negative selection marker (e.g. Drug).

As used herein, the terms "external activator of regulation of CDL" and "EARC" refer to a mechanism or system that promotes exogenous modification of non-coding or coding DNA transcription or corresponding translation via an activator. Is an example of iNEP including. EARC modification allows a user to stop or inhibit mitotic division of cells containing EARC by removing an inducer from the EARC modified cells that allows transcription and/or translation of EARC modified CDL. For example, an inducible activator-based gene expression system can be operably linked to CDL and used to exogenously control CDL expression or CDL translation, which results in CDL transcription and/or translation. Requires the presence of a drug-induced activator and corresponding inducer. In the absence of an inducer, cell division and/or proliferation will be arrested or inhibited (eg, slowed to normal cell division rate). For example, the CDL Cdk1/CDK1 may be modified to contain a dox-bridge so that expression of the CDL Cdk1/CDK1 and cell division and proliferation are only possible in the presence of the inducer (eg doxycycline).

The term “growth antagonist system” as used herein refers to a natural or engineered compound, the presence of which inhibits (totally or partially) the growth of cells.

As used herein, the term "dox-bridge" expresses rtTA by an endogenous or exogenous promoter (Gossen et al., 1995), which brings transcription of the target region under the control of the TRE, thereby driving the promoter. Refers to the mechanism by which activity is separated from the target transcribed region. As used herein, "rtTA" refers to the reverse tetracycline transactivator of the tetracycline inducible system (Gossen et al., 1995) and "TRE" is the promoter consisting of the TetO operator sequence upstream of the minimal promoter. Refers to. Binding of rtTA to the TRE promoter in the presence of doxycycline increases transcription of loci downstream of the TRE promoter. As long as the permissive or nonpermissive state of transcriptional expression of the target region is controlled by doxycycline, the rtTA sequence can be inserted at the same transcriptional unit as the CDL or at a different location in the genome. dox-crosslinking is an example of EARC.

As used herein, the term “failsafe cell” refers to one or more CDL (eg, at least 2, 3, 4, or 5) with one or more homozygous, heterozygous, Refers to cells containing hemizygous or complex heterozygous ALINK or EARC. Fail-safe cells can include either ALINK or EARC or both ALINK and EARC modifications (eg, ALINK and EARC modifications in different CDLs or single CDLs).

As used herein, the term "failsafe" refers to the characteristic of cells that are unlikely to exhibit uncontrolled (eg, tumorigenic) growth. A cell may be considered "failsafe" if cell growth is under the control of a negative regulator or inducer and genetic mutations are less likely to lose the activity of the system controlling growth. Failsafe volume depends on the number of ALINKs and the number of CDLs targeting ALINKs (eg, cells with homozygous modifications of two different CDLs are cells with heterozygous modifications of a single CDL). It has a larger failsafe volume (eg, it is less likely to lose all of the systems that control growth via genetic mutations) Failsafe properties are further described in Table 3.

The patent or patent application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

These and other features of the present disclosure will become more apparent in the following detailed description with reference to the accompanying drawings.

Expression of cloaking proteins (Cd200 (FIG. 1A), FasL (FIG. 1B), H2-M3 (FIG. 1C) and Cd47 (FIG. 1D)) in the C57BL/6 mouse embryonic stem cell line C2 using immunohistochemistry. The representative figure shown is shown. Splenocytes (Fig. 2A), wt B16 melanoma cells (Fig. 2B), cloaked B16 melanoma cells (Fig. 2C), wt ES cells (Fig. 2D) and cloaked ES cells (Fig. 2) in mixed lymphocyte reaction. 2E) is a flow cytometry plot showing T cell activation using 2E). 3 is a schematic and image showing that cloaked (FIG. 3B) B16F10 cancer cells in an allogeneic model are protected from rejection compared to their WT counterparts (FIG. 3A). Non-cloak cells in C57BL/6 (n=5) and non-cloak cells in FVB/N (n=4) (FIG. 3A); Cloak cells in C57BL/6 (n=5) and FVB/N (n=6). Representative image of cloak cells in Fig. 3B. FIG. 6 is a schematic showing that cloaked embryonic stem cells form tumors in syngeneic B6 mouse recipients (top panel) and FVB allogeneic recipients (bottom panel). 1 is a series of photographs showing allogeneic mice with teratomas formed by subcutaneous injection of cloak C57BL/6 ES cells. Red arrows indicate teratomas. 5A shows teratomas in C3H mice, FIG. 5B shows teratomas in FVB/N mice, and FIG. 5C shows teratomas in CD1 mice. 1 is a schematic series of images showing that animals with cloak tissue are not compromised. FIG. 6 is a series of images of FVB/N mice showing further results showing that animals with cloak cells are not immune compromised. 3 shows transgene expression in clone failsafe containing embryonic stem cells from C57BL/6 mice. FIG. 8A shows FasL expression, FIG. 8B shows Ccl21b expression, and FIG. 8C shows Cd200 expression. 3 shows transgene expression in clone failsafe containing embryonic stem cells from C57BL/6 mice. FIG. 8D shows Cd47 expression, FIG. 8E shows Mfge8 expression, and FIG. 8F shows Spi6 expression. 3 shows transgene expression in clone failsafe containing embryonic stem cells from C57BL/6 mice. FIG. 8G shows H2-M3 expression, and FIG. 8H shows PD-L1 expression. 1 is a series of graphs showing cloaking transgene expression in ES cell clones. Each cloaking transgene is shown in a different color. Concentric circles represent expression levels on the log10 scale. Thick bullets represent 1x expression normalized to a positive control (activated leukocytes isolated from mouse lymphoid organs), the next outer rings represent 10x and 100x expression respectively compared to the positive control. .. The innermost ring is 0.1× expression compared to the positive control. Clones NT2 and 15 (indicated by the red squares) had the highest expression of the cloaking gene. These clones survived in the allogeneic host. FIG. 5 is a graph showing expression of the cloaking transgene among whole genome gene expression level distributions for the whole genome of ES cells. All eight cloaking transgenes in the NT2 cell line and NT2-derived teratomas have expression levels in the top 5% of all genes in the ES cell genome, and five cloaking transgenes in the ES cell genome. It has the highest 1% expression level of all genes. The expression level of the transgene in the NT2 cell line and NT2-derived teratomas was successful in achieving allograft resistance. It is a photograph showing C57BL/6-derived teratomas in FVB/N mice. The transgenic system, NT2, resulted in 9 out of 10 injection sites teratomas. Images were taken 3 months after injection. FIG. 11B is an enlarged view of the teratoma indicated by the arrow in FIG. 11A. FIG. 13 is a graph showing teratoma tumor size in syngeneic (FIG. 12A) and allogeneic (FIG. 12B) mice treated with ganciclovir. FIG. 13 is a series of micrographs showing that cloak embryo stem cells injected into both syngeneic (FIG. 13A) and syngeneic (FIG. 13B) hosts can differentiate into all three cell lineages. Fig. 3 is a photomicrograph showing the formation of all three germ layers in a teratoma formed by subcutaneous injection of cloak ES cells into mice. 14A, 14B and 14C show three germ layers (ec=ectoderm, shown in FIG. 14A; en=endoderm, shown in FIG. 14C; me=mesoderm, shown in FIG. 14B). FIG. 14D shows blood vessels indicated by red arrows, confirming that the tissue is well vascularized. 1 is a schematic diagram showing the construction of a vector expressing a target gene essential for alloresistance. 1 is a fluorescence micrograph showing the expression of a cloaking transgene-encoded protein in ES cells. 16A shows the expression of PD-L1, FIG. 16B shows the expression of CD200, FIG. 16C shows the expression of CD47, FIG. 16D shows the expression of FasL, and FIG. 16E shows the expression of H2-M3. Expression is shown, FIG. 16F shows the expression of Ccl21, FIG. 16G shows the expression of Mfge8, and FIG. 16H shows the expression of Spi6. FIG. 17 is a micrograph showing that cloaked ES cells have typical ES cell morphology (FIG. 17A) and express the ES cell marker alkaline phosphatase (FIG. 17B). 19 is a fluorescence micrograph showing the expression of markers for pluripotent ES cells (Oct4 (FIG. 18A) and SSEA1 (FIG. 18B)) in cloak ES cells. The inset images in FIGS. 18A-18B show single channel images of fluorescence micrographs for ES cell markers (Oct4 and SSEA) and DAPI, which labeled nuclei and staining for ES cell markers co-localized with cloak cells. It is localized. FIG. 1 is a schematic diagram showing the immune process inhibited by the cloaking transgene (top) and the expression cassette used for expression of the cloaking transgene in ES cells (bottom). 1 is a series of graphs showing the effect of interferon γ (IFNγ) on MHC levels in ES cells. IFNγ increased MHC levels in wild-type ES cells and ES cells overexpressing the wild-type IFNγ receptor IFNγR1, but ES cells overexpressing the dominant negative form of the IFNγ receptor (IFNγR1 d39). Shows that IFNγR1 d39 completely inhibited IFNγ-mediated upregulation of MHC in ES cells without increasing MHC levels in E. coli.

Description of Cells and Methods For example, a method for providing local immunosuppression at the site of implantation using tools and cells such as genetically modified cells when the cells are transplanted into an allogeneic host is featured. A genetically modified cell comprises one or a set of transgenes, each transgene being a gene product that is cytoplasmic, membrane bound or locally acting, the function of which is that of the host immune system (eg, It encodes a gene product that is to alleviate graft attack leukocytes and NK cell activation) or to act as a defense mechanism against leukocyte attack.

Various cytoplasmic, membrane-bound or locally acting immune factors have been found to regulate local immune compartments and local immune populations. PD-L1 (Brown et al., J Immunol. 170:1257-66 (2003: Curiel et al., Nat Med. 9:562-7 (2003); Dong et al., Nat Med. 8:793-800). (2002)), CD47 ((Willingham et al., Proc Natl Acad Sci US A. 109:6662-7 (2012); Liu et al., PLoS One. 10:e0137345 (2015); Demeure et al.,. J Immunol. 164: 2193-9 (2000)), CD200 (Jenmalm et al., J Immunol. 176:191-9 (2006); Cherwinski et al., J Immunol. 174: 1348-56 (2005); Kretz. -Rommel et al., J Immunol. 178:5595-605 (2007)), FasL (O'Connell et al., J Exp Med. 184: 1075-82 (1996); Ju et al., Nature. 373:. 444-8 (1995); Mazar et al., J Biol Chem. 284:22022-8 (2009)) and Spi6 (Medema Proceedings of the National Academia of Sciences of 20 at 15 of the United States of America: 20 of the United States of America). ); Zhang et al., Immunity. 24:451-61 (2006); Soriano et al., Lung Cancer. 77:38-45 (2012)), including their role in immune regulation. Among the many described, we use the expression of one or more of these immunomodulators in allogeneic cells to local immunity in the host to which the cells are administered. It has been discovered that it may provide suppression and/or reduce allo-rejection.

We modified allogeneic cells by the use of specific immunomodulators introduced into the cell or population of cells. The modified cells evade immune rejection through co-regulation of many different local immune pathways. Such genetically engineered cells can be transplanted "off-the-shelf" to many recipients regardless of genetic background without rejection by the recipient's immune system. This immunomodulatory approach overcomes the need for systemic immunosuppression in transplant recipients, which can be dangerous to the recipient. Thus, an immunosuppressive agent can be administered to a patient that receives the modified cells described herein, but the treatment need not include administration of the immunosuppressive agent. This immunomodulatory approach is costly to induce patient-specific iPS cells, engineer regulatory cells, or induce chimeras via hematopoietic cell transplantation (HCT) and also overcome impractical methodologies ..

The cell may be genetically modified to express a set of transgenes, which is a cytoplasmic, membrane-bound or locally acting gene product, the function of which is that of the host immune system. Encodes a gene product that is to alleviate (eg, graft attack leukocytes and NK cell activation) or act as a defense mechanism against an immune response (eg, leukocyte attack). The set of transgenes can be selected from genes that have a role in the gene regulatory pathways described above. Such genes include, but are not limited to, those provided in Table 1.

The CC motif chemokine ligand 21 (Ccl21) is expressed by regional lymph nodes, where Cc121 acts to attract activated antigen presenting cells (APC). This important function provides the opportunity to "reverse" APC migration by overexpressing this gene on transplanted cells. Indeed, some melanomas express Ccl21, recruit CCR7+ cells and are then able to rearrange parts of their tumor stroma as “self”. This leads to interstitial remodeling that directs recruitment and maintenance of Cd4 ⁺ Tregs (Zindl et al., Science. 328:697-8 (2010)). Indeed, expression of Ccl21 on tumors may protect co-transplanted Ccl21 deficient tumor cells from rejection in a syngeneic allograft setting (Shields et al., Science. 328:749-52 (2010)). Ccl21b is a mouse ortholog of human Ccl21.

The amino acid sequences of mouse and human Ccl21 are as follows.
Mouse Ccl21
Human Ccl21

Expression of Cd47 in the umbilical cord may promote the development of hyporesponsive T cells (Avice et al., J Immunol. 167:2459-68 (2001)). Erythrocytes also upregulate Cd47 to avoid activation of dendritic cells due to their lack of "self" presentation (van den Berg et al., Immunity. 43:622-4 (2015)). .. More recently, increased expression of human Cd47 was shown to increase engraftment in pig-to-human hematopoietic cell transplants in a mouse model (Tena et al., Am J Transplant. 14:2713. -22 (2014)).

The amino acid sequences of mouse and human Cd47 are as follows.
Mouse Cd47
Human Cd47

Cd200 is also an important immunomodulatory (sex) molecule, and its increased expression can reduce the severity of allograft rejection, autoimmunity and allergic disease (Gorczynski et al., J Immunol. 172). : 7744-9 (2004)). In vitro, Cd200 APC expression has been shown to suppress the production of interferon gamma (IFN-γ) and cytolytic granules by activated Cd8+ T cells (Misstar et al., J Virol. 86:6246-. 57 (2012)). Most interestingly, Cd200 overexpression increases skin and cardiac allograft survival in mice by promoting Foxp3+ Treg cells (Gorczynski et al., Transplantation. 98:1271-8 (2014)).

The amino acid sequences of mouse and human Cd200 are as follows.
Mouse Cd200
Human Cd200

Spi6 is an endogenous inhibitor of the cytotoxic effector molecule Granzyme B released by activated Cd8+ T cells (Sun et al., J Biol Chem. 272:15443-41 (1997)). Some data indicate that mesenchymal (lineage) stem cells (MSCs) escape immune rejection by upregulating this molecule (El Haddad et al., Blood. 117: 1176-83 (2011). )). The ability of dendritic cells to present antigen to cytotoxic T cells without being killed by themselves via contact-mediated cytotoxicity has recently been demonstrated to be mediated by Spi6 (Lovo et al., J Immunol. 188:1057-63 (2012)). Spi6 is also known as Serpin B9.

The amino acid sequences of mouse Spi6 and human counterpart Serpin B9 are as follows.
Mouse Spi6
Human Serpin B9

Activated cytotoxic Cd8+ can kill target cells by expression of FasL, which binds to FAS receptors and activates caspase-mediated apoptosis in target cells. However, many tumors developed a "counterattack" by upregulating FasL on their surface (Chen et al., J Immunol. 171:1183-91 (2003)). Selective expression of FasL in human and mouse solid tumor vasculature is associated with rare Cd8+ T cell infiltration and FoxP3+ Treg cell dominance (Motz et al. Nat Med. 20:607-15 (2014)). ). Most recently, B lymphocytes have also been shown to kill T helper cells using the expression of FasL in the effector stage of the immune response (Lundy et al., Front Immunol. 6:122 (2015). )). FasL is a mouse ortholog of human FASLG.

The amino acid sequences of mouse FasL and human counterpart FASLG are as follows.
Mouse FasL
Human FASLG

PD-L1 is an important immunomodulatory molecule that binds to programmed cell death (PD-1). PD-1 is expressed on T cells and binds to PD-L1 resulting in T cell anergy (MacDonald et al., J Immunol. 126:1671-5 (1981)).

The amino acid sequences of mouse and human PD-L1 are as follows.
Mouse PD-L1
Human PDL1 (CD274)

Inflammatory environments such as those induced by allografts attract macrophages and inflammatory monocytes, among many other innate immune cells. Milk fat globule epidermal growth factor 8 (Mfge-8) is expressed by a large number of mouse tumors (Neutzner et al., Cancer Res. 67:6777-85 (2007)) and converts monocytes into suppressive M2-like macrophages. Polarization has been shown to contribute to local immunosuppression (Soki et al., J Biol Chem. 289:24560-72 (2014)).

The amino acid sequences of mouse and human MFGE-8 are as follows.
Mouse MFGE8
Human MFGE8

The potent killing potential of NK cells is also very important in graft rejection. NK cells can kill target cells lacking MHC class I molecules and other cells in the inflammatory setting. H2-M3, a mouse homolog of human HLA-G, has recently been shown to have a regulatory effect on NK cells and is licensed to ignore cells lacking a "self molecule" (Andrews et al. , Nat Immunol. 13: 1171-1-7 (2012)). This is believed to be achieved by the binding of the immunosuppressive receptor, HLA-G, on both NK and T cells (Carrosella et al., Adv Immunol. 127:33-144 (2015)). H2-M3 is a mouse ortholog of human HLA-G.

The amino acid sequences of mouse H2-M3 and human counterpart HLA-G are as follows.
Mouse H2-M3
Human HLA-G

One or more of PD-L1, H2-M3, Cd47, Cd200, FasL, Ccl21b, Mfge8 and Spi6A (for example, 1, 2, 3, 4, 5, 5, 6, 7 or 8) A set of transgenes containing all) can be expressed intracellularly. The cell may be a cell that is subject to genome editing, such as, for example, a stem cell or a cell that can be used therapeutically and/or can differentiate into a therapeutic cell type. Stem cells can be, for example, embryonic stem (ES) cells or induced pluripotent stem (iPS) cells. The set of transgenes may include one, two, three, four, five, six, seven or all eight of these genes, or at least one, at least two of these genes. One, at least three, at least four, at least five, at least six, at least seven may be included. The cells can be further genetically modified to express one or more of TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1 and/or IFNγR1 d39. The TGF-β transgene can be modified to express the gene product in a membrane bound form (ie, the gene product is expressed on the cell surface of an allograft) using methods known to those of skill in the art. .. For example, a method of localizing TGF-β to the membrane is to co-express TGF-β with an additional transgene encoding the LRRC32 protein or any other polypeptide that results in localization of TGF-β to the cell membrane. It is to let. This protein anchors TGF-β to the membrane. (Tran DQ et al., Proc Natl Acad Sci USA 106:13445-50 (2009)).

The amino acid sequence of IFNγR1 d39 is as follows.

The gene can be a human gene or a mouse gene. In embodiments, the gene is of the same species as the recipient of the allograft recipient into which the cells are transplanted. In embodiments, the gene is of any species in which the function of the gene is conserved or the designed biological material has the agonist function of its endogenous counterpart. Methods for introducing and expressing these transgenes in cells are described herein and are known to those of skill in the art. Cells expressing these transgenes may be referred to as "cloaked" because of their ability to avoid allo-rejection without systemic immunosuppression and without the need for immunosuppressive drugs.

It is contemplated herein that the population of cells derived from the above cloak cells can also be used to produce local immunosuppression when transplanted at the transplant site of an allogeneic recipient.

Before or after generating the cloaklet cells of the present disclosure, the cells can first be modified to be failsafe cells. Failsafe cells use the cell division locus (CDL) to control cell growth in animal cells. A CDL is a locus, as provided herein, whose transcripts are expressed during cell division. The CDL can be genetically modified to include a gene expression system based on a negative selectable marker and/or an inducible activator, as described herein, which allows the user to add an appropriate inducer or The removal allows growth, ablation, and/or inhibition of the genetically modified cells. Methods for making and using fail-safe cells are described, for example, in WO 2016/141480, the entire teachings of which are incorporated herein by reference. The cells can be first failsafe and then cloaked. Alternatively, the cells can be cloaked first and then failsafe.

The cell can be a vertebrate cell, eg, a mammalian cell such as a human cell or a mouse cell. The cell can also be a vertebrate stem cell, eg, a mammalian stem cell such as a human stem cell or a mouse stem cell. Preferably, the cells or stem cells are susceptible to genetic modification. Preferably, the cells or stem cells are susceptible to genetic modification. Preferably, the cell or stem cell is considered by the user to have therapeutic value, which is used by the cell or stem cell to treat a disease, disorder, defect or injury in a subject in need thereof. Means to get.

In some embodiments, the cells are stem cells or progenitor cells (eg, iPSCs, embryonic stem cells, hematopoietic stem cells, mesenchymal stem cells, endothelial stem cells, epithelial stem cells, adipose stem cells or progenitor cells, reproductive stem cells, lung stem cells or progenitor cells, Breast stem cells, adult olfactory stem cells, hair follicle stem cells, pluripotent stem cells, amniotic stem cells, cord blood stem cells or neural stem cells or progenitor cells). In some embodiments, the stem cells are adult stem cells (eg, somatic stem cells or tissue-specific stem cells). In some embodiments, stem cells or progenitor cells are capable of differentiating (eg, stem cells are totipotent, pluripotent or pluripotent). In some embodiments, the cells are isolated from embryonic or neonatal tissue. In some embodiments, the cells are fibroblasts, monocyte progenitor cells, B cells, exocrine cells, pancreatic progenitor cells, endocrine progenitor cells, hepatoblasts, myoblasts, preadipocytes, progenitor cells, hepatocytes. , Chondrocytes, smooth muscle cells, K562 human erythroid leukemia cell lines, osteocytes, synovial cells, tendon cells, ligament cells, meniscal cells, adipocytes, dendritic cells or natural killer cells. In some embodiments, the cells are myocytes, erythroid megakaryocytes, eosinophils, iPS cells, macrophages, T cells, islet β cells, neurons, cardiomyocytes, blood cells, endocrine precursor cells, exocrine precursor cells, Engineered (eg, converted or differentiated) into duct cells, acinar cells, α cells, β cells, δ cells, PP cells, hepatocytes, bile duct cells or brown adipocytes. In some embodiments, the cells are muscle cells (eg, skeletal, smooth or cardiomyocytes), red blood cell megakaryocytes, eosinophils, iPS cells, macrophages, T cells, islet β cells, neurons, cardiomyocytes, blood. In cells (eg erythrocytes, leukocytes or platelets), endocrine precursor cells, exocrine precursor cells, duct cells, acinar cells, α cells, β cells, δ cells, PP cells, hepatocytes, cholangiocytes or white or brown adipocytes is there. In some embodiments, the cell is a hormone secreting cell (e.g., insulin, oxytocin, endorphin, vasopressin, serotonin, somatostatin, gastrin, secretin, glucagon, thyroid hormone, bombesin, cholecystokinin, testosterone, estrogen or progesterone, renin. , Cells secreting ghrelin, amylin or pancreatic polypeptide), epidermal keratinocytes, epithelial cells (eg exocrine epithelial cells, thyroid epithelial cells, keratinized epithelial cells, gallbladder epithelial cells or cornea, tongue, oral cavity, esophagus, anus) Canal, distal urethral or vaginal surface epithelial cells), renal cells, germ cells, skeletal joint synovial cells, periosteal cells, osteocytes (eg osteoclasts or osteoblasts), perichondrocytes (eg chondroblasts) Cells or chondrocytes), cartilage cells (eg chondrocytes), fibroblasts, endothelial cells, pericardial cells, meningeal cells, keratinocyte precursor cells, keratinocyte stem cells, pericytes, Glial cells, ependymal cells, cells isolated from amnion or placental membrane or serosa cells (eg serosa cells lining body cavities). In some embodiments, the cells are somatic cells. In some embodiments, the cells are derived from skin or other organs, such as heart, brain or spinal cord, liver, lung, kidney, pancreas, bladder, bone marrow, spleen, intestine or stomach. The cell can be from a human or other mammal, such as a rodent, non-human primate, bovine or porcine cell. It is contemplated herein that cloak cells may be useful in cell-based therapies where it may be desirable to avoid allo-rejection at the local transplant site.

In some embodiments, the clonal cells described herein have a host immune response of 1 week or more (eg, 1 week, 2 weeks, 1 month, 2 months, 3 months, 6 months, 1 year, 2 years. Survive in the host for 3 years, 4 years, 5 years or more, eg, over the life of the cell and/or its progeny) without stimulation. The cells maintain expression of the cloaking transgene as long as they survive in the host (eg, cloak cells can be cleared by the host's immune system if the cloaking transgene is no longer expressed). In some embodiments, the cloak cells further express a transgene encoding a protein (eg, a fluorescent protein such as GFP or other detectable marker) that allows the cloak cells to be detected in vivo. To do.

The combination of cloak cells and fail-safe cells can eliminate cells that show an undesired growth rate before or after transplanting the cells into the host, whether such cells are produced locally or not. It is contemplated herein that it may be useful in cell-based therapies, where it may be desirable to avoid allo-rejection at the site of transplantation. The combination of cloaking and failsafe technologies enables local immune defense while the cells provide local immunosuppression, addressing the risk of the recipient developing malignancy.

Methods of Producing Cloak Cells The compositions and methods described herein can be used to reduce allogeneic cell rejection through expression of the cloaking transgene. Extensive methods have been established for the delivery of proteins to mammalian cells and for the stable expression of genes encoding proteins in mammalian cells, which produce the cloak cells described herein. Can be used to

Polynucleotides Encoding Cloaking Proteins or Therapeutic Agents One platform that can be used to achieve therapeutically effective expression of cloaking proteins or therapeutic agents in mammalian cells is the stability of genes encoding cloaking proteins or therapeutic agents. Expression (eg, by integration into the nucleus of a mammalian cell or the mitochondrial genome or by episomal concatemer formation in the nucleus of a mammalian cell). A gene is a polynucleotide that encodes the primary amino acid sequence of the corresponding protein. Genes can be incorporated into vectors to introduce exogenous genes into mammalian cells. Vectors can be introduced into cells by a variety of methods including transformation, transfection, transduction, direct uptake, projectile bombardment and by encapsulation of the vector in liposomes. Examples of suitable methods for transfecting or transforming cells include calcium phosphate precipitation, electroporation, microinjection, infection, lipofection and direct uptake. Such methods are described, for example, in Green et al. , Molecular Cloning: A Laboratory Manual, Fourth Edition (Cold Spring Harbor University Press, New York 2014); and Ausubel et al. , Current Protocols in Molecular Biology (John Wiley & Sons, New York 2015), the disclosures of each of which are incorporated herein by reference.

Cloaking proteins or therapeutic agents can also be introduced into mammalian cells by targeting a vector containing a portion of the gene encoding the cloaking protein or therapeutic agent to cell membrane phospholipids. For example, the vector can be targeted to phospholipids on the extracellular surface of the cell membrane by linking the vector molecule to the VSV-G protein, a viral protein that has an affinity for all cell membrane phospholipids. Such constructs can be produced using methods well known to those of skill in the art.

The recognition and binding of polynucleotides encoding cloaking proteins or therapeutic agents by mammalian RNA polymerase is important for gene expression. As such, it may include sequence elements within the polynucleotide that exhibit high affinity for transcription factors that recruit RNA polymerase and promote assembly of the transcription complex at the transcription start site. Such sequence elements include, for example, mammalian promoters, the sequences of which may be recognized and bound by specific transcription initiation factors and ultimately RNA polymerase.

Polynucleotides suitable for use in the compositions and methods described herein also include polynucleotides encoding cloaking proteins or therapeutic agents downstream of the mammalian promoter. Promoters useful for expressing cloaking proteins or therapeutic agents in mammalian cells include constitutive promoters. Constitutive promoters include the CAG promoter, cytomegalovirus (CMV) promoter, EF1α promoter and PGK promoter. Alternatively, promoters derived from the viral genome can also be used for stable expression of these agents in mammalian cells. Examples of functional viral promoters that can be used to promote mammalian expression of these agents include adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, HSV tk promoter, mouse mammary tumor virus. (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter and Rous sarcoma virus (RSV) promoter.

When a polynucleotide encoding a cloaking protein or therapeutic agent described herein below is incorporated into the nuclear DNA of mammalian cells, transcription of this polynucleotide can be induced by methods known in the art. For example, expression can be induced by exposing mammalian cells to external chemical reagents, such as agents that regulate the binding of transcription factors and/or RNA polymerases to the mammalian promoter and thus regulate gene expression. .. The chemical reagent can serve to facilitate the binding of RNA polymerase and/or transcription factors to the mammalian promoter, eg, by removing the repressor protein bound to the promoter. Alternatively, the chemical reagent may serve to enhance the affinity of the mammalian promoter for RNA polymerase and/or transcription factors such that the transcription rate of genes located downstream of the promoter is increased in the presence of the chemical reagent. .. Examples of chemical reagents that enhance polynucleotide transcription by the above mechanism include tetracycline and doxycycline. These reagents are commercially available (Life Technologies, Carlsbad, CA) and can be administered to mammalian cells to promote gene expression according to established protocols.

Other DNA sequence elements that may be included in nucleic acid vectors for use in the compositions and methods described herein include enhancer sequences. Enhancers represent another class of regulatory elements that induce conformational changes in a polynucleotide containing a gene of interest such that the DNA adopts a preferred three-dimensional orientation for binding transcription factors and RNA polymerase at the transcription start site. .. Accordingly, polynucleotides for use in the compositions and methods described herein include polynucleotides that encode cloaking proteins or therapeutic agents and further include mammalian enhancer sequences. Many enhancer sequences are presently known from mammalian genes, examples include those from genes encoding mammalian globin, elastase, albumin, α-fetoprotein and insulin. Enhancers for use in the compositions and methods described herein also include those derived from viral genetic material that can infect eukaryotic cells. Examples include the SV40 enhancer on the late side of the replication origin (bp 100 to 270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancer. Additional enhancer sequences that induce activation of eukaryotic gene transcription are described by Yaniv et al. , Nature 297:17 (1982). Enhancers can be spliced into a vector containing a polynucleotide encoding a cloaking protein or therapeutic agent, eg, at the 5'or 3'positions of the gene. In the preferred orientation, the enhancer is located 5'to the promoter, which in turn is located 5'to the polynucleotide encoding the cloaking protein or therapeutic agent.

The nucleic acid vectors described herein can include a Woodchuck post-transcriptional regulatory element (WPRE). WPRE acts at the transcriptional level by promoting the nuclear export of transcripts and/or increasing the efficiency of polyadenylation of nascent transcripts, thus increasing the total amount of mRNA in the cell. Addition of WPRE to the vector can result in a substantial improvement in the level of transgene expression from several different promoters both in vitro and in vivo.

In some embodiments, nucleic acid vectors for use in the compositions and methods described herein include, for example, reporter sequences that may be useful in validating gene expression in specific cells and tissues. .. Reporter sequences that can be provided in the transgene include β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase and in the art. It includes DNA sequences that encode others known in the art. Reporter sequences, when associated with the regulatory elements that drive their expression, are enzymatic, radiographic, colorimetric, fluorescent or other spectroscopic assays, fluorescence activated cell sorting assays as well as enzyme linked immunosorbent assays (ELISA), radioimmunoassays The signal is detectable by conventional means including immunological assays including (RIA) and immunohistochemistry. For example, if the marker sequence is the LacZ gene, the presence of the vector carrying the signal is detected by an assay for β-galactosidase activity. When the transgene is green fluorescent protein or luciferase, the vector carrying the signal can be visually measured by color or light production in a luminometer.

Techniques for Introducing Transgenes into Cells Transfection Techniques that can be used to introduce transgenes, such as the cloaking or therapeutic transgenes described herein, into target cells (eg, mammalian cells). Are well known in the art. For example, electroporation can be used to permeabilize mammalian cells (eg, human target cells) by applying an electrostatic potential to the cells of interest. Mammalian cells, such as human cells, thus exposed to an external electric field are then predisposed to uptake of exogenous nucleic acids. Electroporation of mammalian cells is described, for example, by Chu et al. , Nucleic Acids Research 15:1311 (1987), the disclosure of which is incorporated herein by reference. A similar technique, Nucleofection™, utilizes an applied electric field to stimulate the uptake of exogenous polynucleotides into the nucleus of eukaryotic cells. Nucleofection™ and protocols useful for practicing this technique are described, for example, in Distler et al. , Experimental Dermatology 14:315 (2005) and US Patent Application Publication No. 2010/0317114, the disclosures of each of which are incorporated herein by reference.

Additional techniques useful for transfecting target cells include squeeze poration. This technique induces rapid mechanical deformation of cells in order to stimulate the uptake of exogenous DNA through membranous pores formed in response to applied stress. This technique is advantageous in that the vector is not required for delivery of the nucleic acid to cells such as human target cells. Squeeze poration is described, for example, in Sharei et al. , Journal of Visualized Experiments 81:e50980 (2013), the disclosure of which is incorporated herein by reference.

Lipofection represents another technique useful for transfection of target cells. This method involves the loading of nucleic acids into liposomes, which often present cationic functional groups (eg, quaternary or protonated amines) towards the exterior of the liposome. This facilitates electrostatic interactions between liposomes and cells due to the anionic nature of the cell membrane, which is ultimately due to, for example, direct fusion of the liposomes with the cell membrane or endocytosis of the complex. Leads to uptake of exogenous nucleic acid. Lipofection is described in detail, for example, in US Pat. No. 7,442,386, the disclosure of which is incorporated herein by reference. Similar techniques that utilize ionic interactions with cell membranes to induce uptake of exogenous nucleic acids include contacting cells with cationic polymer-nucleic acid complexes. Exemplary cationic molecules that associate with a polynucleotide to impart a positive charge that favors interaction with the cell membrane include activated dendrimers (eg, Dennig, Topics in Current Chemistry 228:227 (2003) (the disclosure of which is herein incorporated by reference). , Herein incorporated by reference)), polyethyleneimine and diethylaminoethyl (DEAE)-dextran, the use of which as transfection agents is described, for example, in Gulick et al. , Current Protocols in Molecular Biology 40:I:9.2:9.2.1 (1997), the disclosure of which is incorporated herein by reference. Magnetic beads are another tool that can be used to transfect target cells in a mild and efficient manner as the method utilizes an applied magnetic field to guide the uptake of nucleic acids. This technique is described in detail, for example, in US Patent Application Publication No. 2010/0227406, the disclosure of which is incorporated herein by reference.

Another useful tool for inducing the uptake of exogenous nucleic acids by target cells is to gently permeabilize the cells and allow them to penetrate the cell membrane at a specific wavelength of electromagnetic radiation. Laser transfection, also called optical transfection, is a technique that involves exposure to radiation. The bioactivity of this technique is similar to electroporation and is sometimes found to be superior to electroporation.

Impalefection is another technique that can be used to deliver genetic material to target cells. Imperfection depends on the use of nanomaterials such as carbon nanofibers, carbon nanotubes and nanowires. The acicular nanostructures are synthesized perpendicular to the surface of the substrate. DNA containing genes intended for intracellular delivery are attached to the nanostructured surface. The tip with the array of these needles is then pressed against the cell or tissue. Cells that are disturbed by the nanostructures can express the delivered gene. An example of this technique is described by Shalek et al. , PNAS 107:1870 (2010), the disclosure of which is incorporated herein by reference.

Magnetofection can also be used to deliver nucleic acids to target cells. The principle of magnetofection is to associate nucleic acids with cationic magnetic nanoparticles. Magnetic nanoparticles are made from iron oxide, which is completely biodegradable, and coated with unique, cationically specific molecules that vary depending on the application. Their association with gene vectors (DNA, siRNA, viral vectors, etc.) is achieved by salt-induced colloidal aggregation and electrostatic interactions. The magnetic particles are then concentrated on the target cells under the influence of the external magnetic field generated by the magnet. This technique is described in detail in Scherer et al, Gene Therapy 9:102 (2002), the disclosure of which is incorporated herein by reference.

Another useful tool for inducing the uptake of exogenous nucleic acids by target cells is sonoporation, a technique used to modify the permeability (typically ultra Sonic frequency), which makes the cells permeable and allows the polynucleotide to penetrate cell membranes. This technique is described, for example, in Rhodes et al. , Methods in Cell Biology 82:309 (2007), the disclosure of which is incorporated herein by reference.

Microvesicles represent another potential vehicle that can be used to modify the genome of target cells according to the methods described herein. For example, microvesicles induced by co-overexpression of glycoprotein VSV-G and a genomically modified protein, such as a nuclease, are used to efficiently deliver the protein intracellularly and then to the endogenous polynucleotide sequence. Can catalyze the site-specific cleavage of <RTIgt;of,</RTI> to prepare the cell's genome for covalent integration of the polynucleotide of interest, such as genes or regulatory sequences. The use of such vesicles, also called gesicles, for the genetic modification of eukaryotic cells is described, for example, by Quinn et al. , Genetic Modification of Target Cells by Direct Delivery of Active Protein [Summary]: Methylation changes in early embryonic genes in cancer [Summary]: Proceedings of the 18th Annual Meeting of the American Society of Gene and Cell Therapy; 2015 May 13, Abstract No ． 122.

Viral Infection In addition to achieving high rates of transcription and translation, stable expression of exogenous genes in mammalian cells can be achieved by integration of the polynucleotide containing the gene into the mammalian cell's nuclear genome. Various vectors have been developed to deliver and integrate polynucleotides encoding exogenous proteins into the nuclear DNA of mammalian cells. Examples of expression vectors are disclosed, for example, in WO 1994/011026, which is incorporated herein by reference. Expression vectors for use in the compositions and methods described herein include cloaking or therapeutic transgenes and, for example, the expression of these agents and/or their polynucleotide sequences into the genome of mammalian cells. It contains additional sequence elements used for integration. Specific vectors that can be used for expression of the cloaking transgene or the therapeutic transgene include plasmids containing regulatory sequences such as promoter and enhancer regions that direct gene transcription. Other useful vectors for the expression of cloaking transgenes or therapeutic transgenes are polynucleotides that enhance the rate of translation of these genes or improve the stability or export of mRNAs resulting from gene transcription to the nucleus. Contains the sequence. These sequence elements include, for example, 5'and 3'untranslated regions and polyadenylation signal sites to direct the efficient transcription of the genes carried on the expression vector. Expression vectors suitable for use with the compositions and methods described herein can also include a polynucleotide encoding a marker for selection of cells containing such vectors. Examples of suitable markers include genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin or nourseosricin.

Viral Vectors for Nucleic Acid Delivery The viral genome is a rich source of vectors that can be used for efficient delivery of a gene of interest into the genome of a target cell (eg, a mammalian cell such as a human cell). I will provide a. Viral genomes are particularly useful vectors for gene delivery because the polynucleotides contained within such genomes have been integrated into the nuclear genome of mammalian cells by generalized or specialized transduction. This is because it is typically incorporated. These processes occur as part of the natural viral replication cycle and do not require added proteins or reagents to induce gene integration. Examples of viral vectors include retroviruses (eg, retroviridae virus vectors), adenoviruses (eg, Ad5, Ad26, Ad34, Ad35 and Ad48), parvoviruses (eg, adeno-associated virus), coronaviruses, orthomyxos. Minus-strand RNA viruses such as viruses (eg influenza virus), rhabdoviruses (eg rabies and vesicular stomatitis virus), paramyxoviruses (eg measles and Sendai), plus-strand RNA viruses such as picornavirus and alphavirus. And adenoviruses, herpesviruses (eg, herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus) and poxviruses (eg, vaccinia, modified vaccinia ankara (MVA), fowlpox and canalipox). Double-stranded DNA viruses can be mentioned. Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, human papillomavirus, human foam virus and hepatitis virus. Examples of retroviruses include avian leukemia sarcoma, avian C virus, mammalian C virus, B virus, D virus, oncoretrovirus, HTLV-BLV group, lentivirus, alpharetrovirus, gammaretrovirus, spuma. Viruses (Coffin, JM, Retroviridae: The viruses and their replication, Virology, Third Edition (Lippincott-Raven, Philadelphia, 1996)). Other examples include mouse leukemia virus, mouse sarcoma virus, mouse mammary tumor virus, bovine leukemia virus, feline leukemia virus, feline sarcoma virus, avian leukemia virus, human T cell leukemia virus, baboon endogenous virus, gibbon ape leukemia virus, Mason Pfizer Simian Virus, Simian Immunodeficiency Virus, Simian Sarcoma Virus, Rous Sarcoma Virus and Lentivirus. Other examples of vectors are described, for example, in US Pat. No. 5,801,030, the disclosure of which relates to viral vectors for use in gene therapy and is hereby incorporated by reference. Incorporated.

AAV Vectors for Nucleic Acid Delivery In some embodiments, the cloaking transgenes or therapeutic transgenes described herein are incorporated into rAAV vectors and/or virions to facilitate their introduction into cells. Incorporated. RAAV vectors useful in the compositions and methods described herein include (1) promoters, (2) heterologous sequences to be expressed (eg, cloaking transgenes or therapeutic transgenes described herein). And (3) a recombinant nucleic acid construct comprising a viral sequence that facilitates integration and expression of a heterologous gene. Viral sequences can include sequences of AAV required in cis for replication of DNA into virions and packaging (eg, functional ITRs). Such rAAV vectors may also include a marker or reporter gene. Useful rAAV vectors have one or more AAV WT genes that are wholly or partially deleted, but retain functional flanking ITR sequences. The AAV ITR can be of any serotype suitable for a particular application. This method for using rAAV vectors is described, for example, in Tal et al. , J Biomed. Sci. 7:279 (2000) and Monahan and Samulski, Gene Delivery 7:24 (2000) (the disclosure of each of which is incorporated herein by reference as it relates to AAV vectors for gene delivery). Has been done.

The transgenes and vectors (eg, promoters operably linked to a cloaking transgene or therapeutic transgene) described herein include rAAV virions to facilitate introduction of the polynucleotide or vector into cells. Can be incorporated. The AAV capsid protein constitutes the outer non-nucleic acid portion of the virion and is encoded by the AAV cap gene. The cap gene encodes three viral coat proteins required for virion assembly, VP1, VP2 and VP3. Construction of rAAV virions is described, for example, in US Pat. No. 5,173,414; US Pat. No. 5,139,941; US Pat. No. 5,863,541; US Pat. , 305; U.S. Patent No. 6,057,152; and U.S. Patent No. 6,376,237; and Rabinowitz et al. , J. Virol. 76:791 (2002) and Bowles et al. , J. Virol. 77:423 (2003), the disclosure of each of which is incorporated herein by reference as it relates to AAV vectors for gene delivery.

Useful rAAV virions in conjunction with the compositions and methods described herein include AAV 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, rh10, rh39, rh43 and rh74. Included are those from various AAV serotypes. Construction and use of different serotypes of AAV vectors and AAV proteins is described, for example, in Chao et al. , Mol. Ther. 2:619 (2000); Davidson et al, Proc. Natl. Acad. Sci. USA 97:3428 (2000); Xiao et al. , J. Virol. 72:2224 (1998); Halbert et al, J. Am. Virol. 74:1524 (2000); Halbert et al. Virol. 75:6615 (2001); and Auricchio et al. , Hum. Molec. Genet. 10:3075 (2001), the disclosure of each of which is related to AAV vectors for gene delivery and is hereby incorporated by reference.

Pseudotyped rAAV vectors are also useful in connection with the compositions and methods described herein. Pseudotyped vectors include AAV vectors of a given serotype (eg, AAV9), which include serotypes other than the given serotype (eg, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, etc.). ) Is pseudotyped using the capsid gene derived from. Techniques involving the construction and use of pseudotyped rAAV virions are known in the art, eg Duan et al. , J. Virol. 75:7662 (2001); Halbert et al. Virol. 74:1524 (2000); Zolotukhin et al. , Methods, 28:158 (2002); and Auricchio et al. , Hum. Molec. Genet. 10:3075 (2001).

AAV virions with mutations within the virion capsid can be used to more effectively infect specific cell types than non-mutated capsid virions. For example, a suitable AAV variant may have a ligand insertion mutation to facilitate targeting AAV to a particular cell type. Construction and characterization of AAV capsid variants, including insertion variants, alanine screening variants and epitope tag variants are described in Wu et al. , J. Virol. 74:8635 (2000). Other rAAV virions that can be used in the methods described herein include capsid hybrids produced by molecular growth of the virus and by exon shuffling. For example, Soong et al, Nat. Genet. 25:436 (2000) and Kolman and Stemmer, Nat. Biotechnol. 19:423 (2001).

Genome Editing In addition to the above, various tools have been developed that can be used for integration of the gene of interest into target cells such as mammalian cells. One such present method that can be used to integrate the polynucleotide encoding the target gene into target cells involves the use of transposons. A transposon is a polynucleotide that encodes a transposase enzyme and contains the polynucleotide sequence or gene of interest flanked by 5'and 3'excision sites. When the transposon is delivered intracellularly, expression of the transposase gene begins, resulting in an active enzyme that cleaves the gene of interest from the transposon. This activity is mediated by site-specific recognition of transposon excision sites by transposase. In some examples, these excision sites can be terminal repeats or inverted terminal repeats. Once excised from the transposon, the gene of interest can integrate into the mammalian cell's genome by transposase-catalyzed cleavage of similar excision sites present in the cell's nuclear genome. This allows the gene of interest to be inserted into the nuclear DNA cleaved at the complementary excision site, followed by covalent ligation of phosphodiester bonds linking the gene of interest to DNA of the mammalian cell genome. Complete the capture process. In certain cases, the transposon may be a retrotransposon in which the gene encoding the target gene is first transcribed into an RNA product and then reverse transcribed into DNA before being integrated into the mammalian cell genome. Exemplary transposon systems include piggyback transposons (eg, described in detail in WO 2010/085699) and beauty transposons during sleep (eg, described in US 2005/0112764). , Each of which is incorporated herein by reference, as the disclosure of each of these relates to transposons for use in gene delivery to cells of interest.

Another tool for the integration of target genes into the genome of target cells is the clustered, regularly spaced short palindromic repeat (CRISPR)/Cas system, which is used for bacterial and viral infections against viral infections. It is the first system that has evolved as an adaptive defense mechanism in archaea. The CRISPR/Cas system contains palindromic repeats within plasmid DNA and associated Cas9 nuclease. This ensemble of DNAs and proteins directs site-specific DNA cleavage of target sequences by first incorporating foreign DNA into the CRISPR locus. Polynucleotides containing these foreign sequences and repeating spacer elements of the CRISPR locus are then transcribed in the host cell to produce guide RNA, which then anneals to the target sequence and the Cas9 nuclease at this site. Can be localized. In this way, highly site-specific cas9-mediated DNA cleavage can be triggered in the foreign polynucleotide, since the interaction that brings cas9 into close proximity to the target DNA molecule is governed by RNA:DNA hybridization. Is. As a result, a CRISPR/Cas system can be designed to cleave any target DNA molecule of interest. This technique has been used to edit eukaryotic genomes (Hwang et al., Nature Biotechnology 31:227 (2013)) and to cleave DNA prior to integration of the gene encoding the target gene, It can be used as an efficient means of site-specific editing of the target cell genome. The use of CRISPR/Cas to regulate gene expression is described, for example, in US Pat. No. 8,697,359, the disclosure of which relates to the use of the CRISPR/Cas system for genome editing. Are incorporated herein by reference. An alternative method for site-specific cleavage of genomic DNA prior to integration of the gene of interest into target cells involves the use of zinc finger nuclease (ZFN) and transcriptional activator-like effector nuclease (TALEN). Unlike the CRISPR/Cas system, these enzymes do not contain an inducing polynucleotide to localize to a particular target sequence. Instead, target specificity is controlled by the DNA binding domain within these enzymes. The use of ZFN and TALEN in genome editing applications is described, for example, by Urnov et al. , Nature Reviews Genetics 11:636 (2010); and Jung et al. , Nature Reviews Molecular Cell Biology 14:49 (2013), the disclosure of each of which is incorporated herein by reference as they relate to compositions and methods for genome editing.

A further genome editing technique that can be used to integrate the polynucleotide encoding the target gene into the genome of the target cell is the use of ARCUS™ meganuclease, which can be rationally designed to cleave genomic DNA in a site-specific manner. including. The use of these enzymes to integrate the gene encoding the target gene into the genome of mammalian cells is advantageous in view of the well-defined structure-activity relationships established for such enzymes. Single-stranded meganucleases are modified at specific amino acid positions to selectively cleave DNA at desired positions, creating nucleases that allow site-specific integration of target genes into the nuclear DNA of target cells. obtain. These single-stranded nucleases are extensively described in, for example, US Pat. No. 8,021,867 and US Pat. No. 8,445,251, the disclosures of each of which are Incorporated herein by reference as it relates to compositions and methods therefor.

Expression of Cloaking Transgenes The cloaking transgenes described herein (eg PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 ( One or any combination of Spi6) prevents rejection when injected into a subject (eg a mammalian subject such as a mouse, rat or human) in an amount sufficient to produce a cloaking effect. Is expressed in an amount sufficient to Transgene expression may be thought to produce a cloaking effect if subcutaneous injection of cloak cells produces teratomas that are not cleared by the subject's immune system. The cloaking transgene is also expressed at levels sufficient to facilitate production of the protein encoded by said transgene. Protein production can be detected using routine methods known to those of skill in the art, such as immunohistochemistry, Western blot analysis or visualization or other methods that allow proteins. Preferably, expression of the cloaking transgene is that all eight proteins encoded by the cloaking transgene (PD-L1, H2-M3, Cd47, Cd200, FasL, Ccl21b, Mfge8 and Spi6) can be detected in cloak cells. As such (eg, detected by immunohistochemistry using antibodies to the protein encoded by the cloaking transgene).

In some embodiments, the cloaking transgene is expressed in cloak cells at levels similar to those of endogenous gene expression in activated leukocytes such as T cells (eg, isolated from lymphoid organs. Activated leukocytes from the same species, such as activated leukocytes, eg expression in cloaked mouse cells is similar to that in activated leukocytes isolated from mouse lymphoid organs). One or more cloaking transgenes (e.g. one of PD-Ll, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6), 2; Expression of one, three, four, five, six, seven or eight) is greater than or equal to expression of an endogenous gene in activated leukocytes (eg, T cells) from the same species (eg, cloaking). The expression level of the transgene is equal to the expression level of the endogenous gene in activated leukocytes, or 1.5, 2, 3, 4, 5, 6, 7, than the expression level of the endogenous gene in activated leukocytes. 8, 9, 10 times higher). In some embodiments, all eight cloaking transgenes are expressed at or above the level of expression of an endogenous gene in activated leukocytes from the same species. Activated leukocytes can be isolated from lymphoid organs, or leukocytes such as T cells can be activated in vitro with anti-CD3/CD28 beads or other methods used by those skilled in the art ( See, for example, Frauwith and Thompson, J. Clin Invest 109:295-299 (2002); and Trickett and Kwan, J Immunol Methods 275:251-255 (2003). Transgene expression in cloak cells can also be compared to gene expression levels reported in profiling studies of activated T cells (see, eg, Palacios et al., PLOSone 2:e1222 (2007)). In some embodiments, cloaking transgene expression is compared to expression of an endogenous gene in a wild-type version of the cell (eg, a stem cell, eg, an embryonic stem cell from the same species as the cloak cell). One or more cloaking transgenes (e.g. one of PD-Ll, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6), 2; Expression in unmodified wild-type cells of the same cell type as the cloak cells (eg stem cells, eg embryonic stem cells from the same species). At least 2,3,4,5,6,7,8,9,10,20,30,40,50,100,500,1,000 or more times higher in cloak cells compared to the expression of the endogenous gene. In some embodiments, all eight cloaking transgenes have levels (eg, 2 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 or 100 or more times higher). Gene expression can be assessed by direct comparison with isolated ES cells or by comparison with stem cell expression (eg, ES cell expression) in the Project Grandiose dataset (www.stemformatics.org/project_grandiose). Gene expression can be measured using techniques known in the art, such as quantitative polymerase chain reaction (qPCR).

Methods of Providing Local Immunosuppression at the Site of Transplant In addition, methods of providing local immunosuppression at the site of transplant are also featured.

The method comprises providing a cell and expressing a set of transgenes in the cell, wherein each transgene is a gene product that is cytoplasmic, membrane bound or locally acting, and whose function is Encodes a gene product that is to mitigate the function of graft-attacking leukocytes and NK cell activation, or to act as a defense mechanism against leukocyte attack.

The set of transgenes includes one or more of the following genes: PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6). For example, two, three, four, five, six, seven or all eight). In an embodiment, the set of transgene genes comprises Pd-L1, H2-M3, Cd47, Cd200, FasL, Ccl21b, Mfge8 and Spi6.

Optionally, the method comprises expressing in a cell one or more of the following transgenes: TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1 and IFNγR1 d39. Further includes. In embodiments, the TGF-β or biological agent is locally acting.

Techniques for introducing various genetic modifications such as transgenes into animal cells are described herein and are generally known in the art.

In an embodiment of the method, the cells are stem cells, cells that are subject to genome editing and/or a source of therapeutic cell types (eg, cells that can be differentiated into lineage-restricted cells for cell therapy or cells of the desired target tissue). is there. In an embodiment, the cells are embryonic stem cells, induced pluripotent stem cells, adult stem cells, tissue-specific stem cells, hematopoietic stem cells, mesenchymal stem cells, endothelial stem cells, epithelial stem cells, adipose stem cells or progenitor cells, germ stem cells, lung stem cells or progenitors. Cells, breast stem cells, adult olfactory stem cells, hair follicle stem cells, pluripotent stem cells, amniotic stem cells, cord blood stem cells or neural stem cells or progenitor cells. In some embodiments, the cells are derived from a target tissue, such as skin, heart, brain or spinal cord, liver, lung, kidney, pancreas, bladder, bone marrow, spleen, intestine or stomach. In some embodiments, the cells are fibroblasts, epithelial cells or endothelial cells. The cell can be a vertebrate cell, eg, a mammalian cell such as a human or mouse cell. In some embodiments, one or more of PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6) (eg, 2 One, three, four, five, six, seven or all eight) cells modified within the tissue or organ to be transplanted. In some embodiments, cloak cells (eg, cloak stem cells) are differentiated into tissues or organs for transplantation in vitro using methods known to those of skill in the art.

In some embodiments, between 1 and 100 billion cloak cells (eg, 1×10 ⁶ , 2×10 ⁶ , 3×10 ⁶ , 4×10 ⁶ , 5×10 ⁶ , 6×10 ⁶ , 7). X10 ⁶ , 8x10 ⁶ , 9x10 ⁶ , 1x10 ⁷ , 2x10 ⁷ , 3x10 ⁷ , 4x10 ⁷ , 5x10 ⁷ , 6x10 ⁷ , 7x10 ⁷ , 8 X10 ⁷ , 9x10 ⁷ , 1x10 ⁸ , 2x10 ⁸ , 3x10 ⁸ , 4x10 ⁸ , 5x10 ⁸ , 6x10 ⁸ , 7x10 ⁸ , 8x10 ⁸ , 9 ×10 ⁸ , 1×10 ⁹ , 2×10 ⁹ , 3×10 ⁹ , 4×10 ⁹ , 5×10 ⁹ , 6×10 ⁹ , 7×10 ⁹ , 8×10 ⁹ , 9×10 ⁹ , 1 ×10 ¹⁰ , 2×10 ¹⁰ , 3×10 ¹⁰ , 4×10 ¹⁰ , 5×10 ¹⁰ , 6×10 ¹⁰ , 7×10 ¹⁰ , 8×10 ¹⁰ , 9×10 ¹⁰ or 1×10 ¹¹ Cloak cells) are administered within or near the site of transplantation in the subject or into the organ or tissue to be transplanted.

Techniques for transplanting genetically modified cells into the transplant site of an allogeneic host are described herein and are known in the art.

Expression of Therapeutic Agents by Cloak Cells The cloak cells described herein can be further modified to express a therapeutic agent. In some embodiments, the therapeutic agent is a protein. A therapeutic protein is a wild-type form of a protein that is deficient in a subject, such as a protein that has been mutated by a cell of the subject or that is produced in insufficient amounts (eg, produced at low levels or not). Can be In some embodiments, the therapeutic protein is an inhibitory antibody (eg, an antibody that blocks or neutralizes protein function). Cloak cells are diseases or conditions associated with overproduction or aberrant production of protein (eg, production by cells that normally do not produce protein, production of protein at times or locations where protein is not normally produced, or production of excess protein). It can be modified to produce inhibitory antibodies for treating subjects who have or are at risk of developing a condition. In some embodiments, the therapeutic antibody is an agonist antibody (eg, activating antibody). Agonist antibodies bind to and activate endogenous receptors (e.g., induce or increase signaling downstream of receptor activation, or open conformation of the endogenous receptor or By changing to the active state). Cloak cells can be modified to produce agonist antibodies to treat subjects who have or are at risk of developing a disease or condition associated with under-activation of receptors or signaling pathways. Cloak cells can be modified to produce therapeutic proteins or antibodies using the methods described herein or other methods known to those of skill in the art. Cloak cells that produce secreted proteins or antibodies can be delivered as circulating cells and can be injected into tissues, organs or body parts in need of therapeutic proteins or antibodies, or subcutaneously to produce cloaked subcutaneous tissue. Can be injected. Cloak cells producing transmembrane or membrane-bound proteins can be injected at or near the site of endogenous cells that respond to the therapeutic protein.

In some embodiments, the cloak cells described herein provide a wild-type copy of the gene that is mutated in the subject (eg, cloak cells have no genetic cause of the disease, PD -L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6) 1, 2, 3, 4, 5, 6 “Wild-type cells” that express one, seven, or all eight). Such cells treat a subject having a disease or condition caused by a mutation in an endogenous gene (eg, a subject having a metabolic disorder associated with one or more mutations described herein below). Can be used for.

A list of exemplary therapeutic agents that can be administered with or produced by cloak cells and related diseases or conditions that can be treated using these therapeutic agents is provided in Table 2 below.

Inducible Systems for Expression of Therapeutic Agents Where continuous administration of therapeutic agents expressed by cloak cells is required to treat a disease or condition, the therapeutic agents are described herein or are presently known. It can be expressed using a constitutive promoter known to those of skill in the art, such as CAG, CMV or another constitutive promoter. If the therapeutic agent is needed intermittently (eg, during the period of relapse or relapse that occurs during a disease or condition, but not if the subject is asymptomatic), the therapeutic agent is , Can be expressed by an inducible promoter that provides the ability to express the therapeutic agent only when needed. One exemplary class of therapeutic agents that can be delivered using inducible promoters are TNFα inhibitors. TNFα inhibitors are currently used to treat rheumatoid arthritis, but constitutive administration of TNFα can result in systemic immunosuppression and is therefore given intermittently during exacerbation of joint inflammation. Nothing more than. If the cloak cells are modified to express a TNFα inhibitor under the control of an inducible promoter, the cloak cells can be used to deliver TNFα intermittently, thus eliminating the need for repeated infusions. Other therapeutic agents that have potentially deleterious effects when administered sequentially can also be expressed intermittently using inducible promoters as described herein. An exemplary inducible expression system is described below.

Tetracycline Response Element One widely used inducible expression system is based on tetracycline-controlled transcriptional activation. In this system, the antibiotic tetracycline or one of its derivatives (eg doxycycline) is used to reversibly activate or inhibit gene expression. In order to use this system, the tetracycline response element (TRE) is a minimal promoter that typically has a very low basal expression upstream of the gene of interest (eg, the therapeutic transgene to be expressed by cloak cells). It is arranged with. A protein called rtTA, which also needs to be expressed by clonal cells, binds TRE and activates transcription in the presence of tetracycline or doxycycline. When tetracycline or doxycycline is removed, rtTA no longer binds to TRE and the gene of interest is no longer expressed. The system Tet-On Advanced transactivator (rtTA2 ^s -M2) and Tet-On 3G advances a version is human codon optimized for responding to low concentrations of doxycycline may be particularly useful in human therapy ..

Light-Induced System Another method for inducible activation of gene expression involves the use of optogenetics to engineer gene expression with photosensitive proteins. Recent developments in optogenetics that can be used to inducibly express therapeutic agents in cloak cells include a class of proteins that undergo conformational changes and dimerization in response to blue light. These proteins are fused to specific promoter sequences and to DNA-binding and transcription components that have been shown to activate transcription when bound by exposure to blue light (Wang et al. ,, Nat Methods, 9:266-269, 2012). Since blue light can irradiate the skin near cloaked subcutaneous tissue to induce the production of therapeutic agents by cloak cells, a method of inducibly activating gene expression is in cloak cells administered subcutaneously. It can be used to control the production of therapeutic agents.

Radiogenetics A third method of inducibly activating gene expression (eg, expression of therapeutic agents by cloak cells) involves the use of radio waves. In one version of the radio-inducible expression system, the TRPV1 receptor is co-expressed with a form of ferritin fused to the GFP binding domain and linked to GFP (Stanley et al., Nat Med 21:92-98, 2015). GFP-ferritin binds to the GFP binding domain of the TRPV1 receptor. When radio waves of a specific frequency are applied to cells, ferritin interacts with TRPV1 and allows calcium influx, which activates the transcription factor NFAT. Therapeutic agents can be inducibly expressed using this system when operably linked to a NFAT-sensitive promoter element (eg, SRE-CRE-NFATRE) and co-expressed with TRPV1-GFP and GFP-ferritin. .. Radio-induced expression offers the advantage that radio waves can pass through tissues and thus can be induced in cells further away from the outside of the body. For example, radiogenetics can be used to regulate gene expression in the retina. Therefore, the method can be used to inducibly express therapeutic transgenes in cloak cells using non-invasive and non-harmful radio waves.

Destabilization Domain System Gene expression can also be regulated using a destabilization domain system. A transgene encoding a protein of interest (eg, a therapeutic agent described herein) may also include a destabilization domain, such that the resulting protein product is fused to the destabilization domain. Contains protein. Exemplary destabilization domains include variants of human FK506 and rapamycin binding protein (FKBP12), which impart instability to the proteins to which they are fused. FKBP12 mutants include N-terminal mutants F15S, V24A, H25R, E60G and L106P and C-terminal mutants M66T, R71G, D100G, D100N, E102G and K105I, which are described in Banaszynski et al. , Cell 126:995 (2006), the disclosure of which is related to the FKBP12 destabilization domain and is hereby incorporated by reference. The destabilization domain promotes protein degradation. If expression of the protein of interest (eg, therapeutic agent) is desired, small molecule synthetic ligands can be used to stabilize the destabilizing domain-containing protein. The small molecule ligand Shield-1 (Shld1) can be used to stabilize FKBP12 mutant-containing proteins by protecting them from degradation. Other destabilizing domains that can be used to regulate the expressed protein of interest include mutants of E. coli dihydrofolate reductase (ecDHFR) and mutants of the human estrogen receptor ligand binding domain (ERLBD). , Which impart instability that results in degradation when fused to the protein of interest, and is described by Iwamoto et al. , Chem Biol. 17:981 (2010) and Miyazaki et al. , J Am Chem Soc. , 134:3942 (2012), the disclosure of each of which is related to the destabilizing domain system and is hereby incorporated by reference, by the small molecule ligand trimethoprim (TMP), respectively. Alternatively, it can be stabilized with CMP8 or 4-hydroxy tamoxifen (4OHT).

The Kumate Switch Induction System Another method for inducible activation of gene expression involves the use of the cumate gene switch system. In the repressor configuration of this system, regulation is mediated by binding of the repressor (CymR) to an operator site (CuO) located downstream of a strong constitutive promoter. Addition of the small molecule, cumate, relieves repression and allows transgene expression. Alternatively, a reverse cumate transactivator (rcTA) can be inserted upstream of the minimal CMV promoter operably linked to the transgene encoding the therapeutic agent. Six repeats of the cumate operator (6xCuO) can be inserted just before the start of translation (ATG) of the therapeutic transgene. In the absence of cumate, rcTA cannot bind to 6xCuO, so the transgene encoding the therapeutic is not transcribed because 6xCuO is not active. When cumate is added, it forms a complex with rcTA, which allows binding to 6xCuO and transcription of the transgene encoding the therapeutic agent (Mullick et al., 2006).

Ecdysone Induction System Another example of an inducible gene expression system is the N-terminal truncation of the ecdysone receptor (EcR) fused to the activation domains of retinoid X receptor (RXR) and Vp16 (VpEcR) simultaneously by an endogenous promoter. It is an ecdysone inducible system that is inserted into the 5'untranslated region of genes expressed by cloak cells so that they are expressed. An ecdysone response element (EcRE) with a downstream minimal promoter can be inserted directly upstream of the start codon of the transgene encoding the therapeutic agent. The co-expressed RXR and VpEcR may heterodimerize with each other. In the absence of ecdysone or the synthetic drug analog muristerone A, the dimerized RXR/VpEcR is unable to bind EcRE and thus the transgene encoding the therapeutic agent is not transcribed. In the presence of ecdysone or muristerone A, the dimerized RXR/VpEcR can bind EcRE, thus transcribing the transgene encoding the therapeutic agent (No et al., 1996). Since ecdysone administration has no apparent effect on mammals, its use to regulate genes should be excellent for transient inducible expression of any gene.

Ligand Reversible Dimerization System In another example, a transgene encoding a therapeutic agent can be modified to be functionally divided into moieties/domains such as the 5′ and 3′ moieties, and the FKBP peptide Sequences can be inserted in each domain. The IRES (Internal Ribosomal Entry Site) sequence can be placed between the two domains, which allows the two different domains to be cotranscribed to produce two distinct proteins. In the absence of the dimerizing agent, the two separate domains of the therapeutic agent are functionally inactive. Upon introduction of a dimerizing agent such as rapamycin or AP20187, the FKBP peptide dimerizes, bringing together the 5'and 3'domains of the therapeutic agent and reconstituting the active protein (Rollins et al., 2000). ..

Cell-Based Delivery of Therapeutic Agents Treatment of Age-Related Macular Degeneration or Retinal Dystrophy In one example, cloak cells are treated with a VEGF trap (eg, herein by reference) to treat age-related macular degeneration (AMD) or retinal dystrophy. Modified to produce a VEGF inhibitor such as the soluble decoy receptors described in Holash et al. obtain. VEGF traps are biological substances that bind to and inhibit VEGF, an angiogenic protein that can promote the formation of abnormal blood vessels. VEGF traps are used to treat wet AMD, which is characterized by abnormal growth of subretinal blood vessels that can lead to retinal detachment and progressive visual loss. To treat AMD, VEGF traps are typically delivered by regular injections into the eye. Cloak cells can be modified to produce a VEGF trap or another VEGF inhibitor by expression of a transgene encoding a VEGF trap or another VEGF inhibitor operably linked to a constitutive or inducible promoter. .. Cloak cells (eg, stem cells) expressing a VEGF inhibitor (eg, VEGF trap) can be differentiated into retinal pigment epithelium (RPE) cells prior to administration to the eye using methods known to those of skill in the art, or Isolated RPE cells can be modified to express the cloaking transgene and VEGF inhibitor. 25,000-100,000 cloaked RPE cells (eg, 25,000, 50,000, 75,000) expressing a VEGF inhibitor (eg, VEGF trap) for treating wet AMD or retinal dystrophy. Or 100,000 cloaked RPE cells) can be injected into the subretinal space of each eye. Other VEGF inhibitors suitable for use in the compositions and methods described herein include soluble forms of the VEGF receptor (eg, soluble VEGFR-1 or NRP-1), platelet factor-4, prolactin, Includes SPARC and VEGF inhibitory antibodies (eg, bevacizumab and ranibizumab).

Treatment of Parkinson's Disease In another example, clonal cells such as dopaminergic neurons or cells that can be differentiated in vitro to produce dopaminergic neurons using methods known to those of skill in the art (eg, stem cells). , Can be administered to a subject suffering from Parkinson's disease characterized by loss of dopaminergic neurons. 25,000 to 100,000 cloaked dopaminergic neurons (eg, 25,000, 50,000, 75,000 or 100,000 cloaked dopaminergic neurons) are affected by Parkinson's disease. Can be administered to the brain of a subject (eg, stereotactically injected into the substantia nigra).

Treatment of Myocardial Infarction The cloak cells described herein can also be used to treat myocardial infarction, such as myocardial infarction commonly known as heart attack. Myocardial infarction occurs when blood flow to a portion of the heart is diminished or stopped, causing damage to the heart muscle. To treat a subject suffering from myocardial infarction, the cloak cells (eg, stem cells) can be differentiated into cardiomyocytes using methods known to those of skill in the art, or the isolated cardiomyocytes can be cloaking transgenes. Can be modified to express 500 to 5 billion cloaked cardiomyocytes (eg 5×10 ⁸ , 6×10 ⁸ , 7×10 ⁸ , 8×10 ⁸ , 9×10 ⁸ , 1×10 ⁹ , 2×10 ⁹ , 3×10 ⁹ , 4×10 ⁹ or 5×10 ⁹ cloaked cardiomyocytes) to treat a subject with myocardial infarction (eg, to replace dead or damaged cardiomyocytes). ) Can be administered to a subject by injection into the myocardium.

Treatment of Osteoarthritis and Rheumatoid Arthritis In another example, the cloak cells described herein can be used to treat osteoarthritis or rheumatoid arthritis. Osteoarthritis and rheumatoid arthritis (RA) are characterized by joint inflammation and are commonly treated with anti-inflammatory therapeutic agents. To treat a subject suffering from osteoarthritis or RA, the cloak cells express an anti-inflammatory biological agent (eg, an inhibitor of TNFα (eg, a TNFα inhibitory antibody)). It can be modified and is already in clinical use for the treatment of RA. Cloak cells produce anti-inflammatory biological agents such as TNFα inhibitors by expression of a transgene encoding an anti-inflammatory biological agent operably linked to a constitutive or inducible promoter. Can be modified to Cloak cells (eg, stem cells) expressing anti-inflammatory biological agents (eg, TNFα inhibitors) can be differentiated into joint fibroblasts prior to administration to the joint using methods known to those of skill in the art. Alternatively, the isolated joint fibroblasts can be modified to express the cloaking transgene and anti-inflammatory biological agents. 1 to 100 million cloak joint fibroblasts expressing anti-inflammatory biological substances (eg 1×10 ⁶ , 2×10 ⁶ , 3×10 ⁶ , 4×10 ⁶ , 5×). 10 ⁶ , 6×10 ⁶ , 7×10 ⁶ , 8×10 ⁶ , 9×10 ⁶ , 1×10 ⁷ , 2×10 ⁷ , 3×10 ⁷ , 4×10 ⁷ , 5×10 ⁷ , 6× 10 ⁷ , 7×10 ⁷ , 8×10 ⁷ , 9×10 ⁷ or 1×10 ⁸ cloak joint fibroblasts) to treat arthritis or inflammatory joints (joints) to treat osteoarthritis or RA. Can be injected). Anti-inflammatory biologicals that can be expressed by cloak cells to treat osteoarthritis or RA include TNFα inhibitors (adalimumab, etanercept, infliximab, golimumab, certrolizumab), interleukin-6 ( IL6) receptor inhibitors (eg tocilizumab), IL1 receptor inhibitors (eg anakinra) or other agents used to treat RA (eg abatacept, rituximab).

Treatment of Diabetes Cloak cells can be used to treat diabetes (eg, type 1 or type 2 diabetes). Type 1 diabetes is due to the inability of the pancreas to produce sufficient insulin. Type 2 diabetes begins with insulin resistance, but insulin deficiency may develop as the disease progresses. To treat a subject suffering from diabetes, the cloak cells can be modified to express insulin, or insulin-expressing cells from a healthy subject (eg, pancreatic β from a subject without diabetes). Cell) is one or more of cloaking transgene PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6) (for example, 1 One, two, three, four, five, six, seven or all eight) and is administered to a subject with diabetes. Cloak cells can be modified to produce insulin by expression of a transgene encoding insulin operably linked to a constitutive or inducible promoter. Can insulin-expressing cloak cells (eg, stem cells) be differentiated into insulin-producing cells (eg, pancreatic β-cells) or administered without differentiation prior to administration using methods known to those of skill in the art? , Or the isolated pancreatic β-cells can be modified to express a cloaking transgene and, optionally, an insulin-encoding transgene. 800 to 3 billion cloaked pancreatic beta cells that express insulin (eg, express insulin endogenously or due to the expression of a transgene encoding insulin) (eg, 8×10 ⁸ , 9×10 ⁸ , 1×10 ⁹ , 2×10 ⁹ or 3×10 ⁹ cloaked pancreatic β-cells), insulin-producing cloaked subcutaneous tissue for treating diabetes. Can be injected subcutaneously into the subject to create a.

Treatment of Hemophilia In another example, the cloak cells described herein can be used to treat hemophilia. Hemophilia patients do not produce functional Factor VIII protein, a key blood component required for blood coagulation. These patients may have severe bleeding and standard care included multiple infusions of purified Factor VIII protein per week. Cloak cells can be modified to express an additional transgene encoding Factor VIII to treat a subject suffering from hemophilia. Factor VIII is constitutively expressed in cloak cells by being operably linked to a constitutive promoter such as CMV or CAG. Do Cloak Cells Expressing Factor VIII (eg Stem Cells) Differentiate into Cells Producing Blood Coagulation Factors (eg Liver Sinusoidal Cells or Endothelial Cells) Prior to Administration Using Methods Known to Those of Skill in the Art? Alternatively, it can be administered without differentiation, or isolated Factor VIII-expressing hepatic sinusoidal cells or endothelial cells from a healthy subject (eg, a subject without haemophilia) are treated with cloaking transgene PD. -L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6) one or more (for example, one, two, three, 4, 5, 6, 7 or all 8) and are administered to a subject with hemophilia. An isolated Factor VIII-expressing hepatic sinusoidal cell or endothelial cell from a healthy subject modified to express one or more cloaking transgenes has a high level of Factor VIII expression. Can be further modified to express the transgene encoding Factor VIII, if desired. 800 to 3 billion cloaks that express Factor VIII (eg, express Factor VIII endogenously or express Factor VIII due to the expression of a transgene encoding Factor VIII). Cloaks that produce Factor VIII to treat cells (eg, 8×10 ⁸ , 9×10 ⁸ , 1×10 ⁹ , 2×10 ⁹ or 3×10 ⁹ cloak cells) for treating hemophilia. The subject can be injected subcutaneously to create a subcutaneous tissue.

Treatment of Metabolic Disorders Cloak cells of the invention can also be used to treat inherited metabolic disorders. In most inherited metabolic disorders, a single enzyme is either not produced in the body or is produced in a defective form. Hereditary metabolic disorders include lysosomal storage disorders such as Hurler syndrome (α-L iduronidase deficiency), Niemann-Pick disease (SMPD1, NPC1 or NPC2 mutation), Tay-Sachs disease (HEXA mutation), Gaucher disease (GBA gene mutation). ), Fabry disease (alpha galactosidase deficiency due to GLA mutation) and Krabbe disease (galactosylceramidase deficiency due to GALC mutation); galactosemia (galactosinase or galactose-1-phosphate ureatransferase deficiency), maple syrup uremia (enzyme). BCKD deficiency); phenylketonuria (enzyme PAH deficiency); glycogen storage disease (GSD), such as GSD0 (glycogen synthase (GYS2) deficiency), GSD1/von Gierke disease (glucose-6-phosphatase (G6PC) deficiency), GSD2/Pompe disease (acidic α-glucosidase (GAA) deficiency), GSD3/Cori disease or Forbes disease (glycogen debranching enzyme AGL deficiency), GSD4/Anderson disease (glycogen branching enzyme (GBE1) deficiency), GSD5 /Mccardle disease (muscle glycogen phosphorylase (PYGM) deficiency), GSD 6/Haas disease (liver glycogen phosphorylase (PYGL) or muscle phosphoglycerate mutase (PGAM2) deficiency), GSD 7/Tarui disease (muscle phosphofructokinase (PMS) Deficiency of PKFM), GSD 9 (deficiency of phosphorylase kinase (PHKA2, PHKB, PHKG2 or PHKA1)), GSD 10 (deficiency of enolase 3 (ENO3)), GSD 11 (deficiency of muscle lactate dehydrogenase (LDHA)), Fanconi Of Bickel syndrome (deficiency of glucose transporter 2 (GLUT2)), GSD 12 (deficiency of aldolase A (ALDOA)), GSD 13 (deficiency of β-enolase (ENO3)) or GSD 15 (glycogenin-1 (GYG1)). Deficiency); mitochondrial diseases such as mitochondrial myopathy (Kerns-Sayer syndrome (KSS, due to mitochondrial DNA deficiency) and chronic progressive external ophthalmoplegia (CPEO, mitochondrial DNA deficiency or replication or ANT1, POLG, POLG2 or PEO1 mutation), diabetes And deafness (DAD, due to mutations in mitochondrial DNA at position 3243 encoding tRNALeu(UUR)), Leber's hereditary optic neuropathy (LHON, MT-ND1, MT-ND4, MT-ND4L and Due to mutations in MT-ND6), Leigh syndrome (SURF1, MT-ATP6, MT-ND2, MT-ND3, MT-ND5, MT-ND6, BCS1L, NDUFA10, SDHA, NDUFS4, NDUFAF2, NDUFA2, NDUFAF6, COX15, ND15. , NDUFS8, FOXRED1, NDUFA9, NDUFA12, associated with mutations in NDUFS7), neuropathy, ataxia, retinitis pigmentosa and ptosis (due to mutations in NARP, MT-ATP6), myouroneuric gastrointestinal encephalopathy. MNGIE, due to mutations in TYMP), myoclonic epilepsy with ragged red fibers (MERRF, sin MT-TK, MT-TL1, MT-TH, MT-TS1, MT-TS2 or MT-TS1 or MT-TS2). Or due to a mutation in mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms (in MELAS, MT-ND1, MT-ND5, MT-TH, MT-TL1 or MT-TV); Friedrich's ataxia ( (Mutations in FXN); peroxisomal diseases such as Zellweger syndrome (mutations in PEX1, PEX2, PEX3, PEX5, PEX6, PEX10, PEX12, PEX13, PEX14, PEX16, PEX19 or PEX26) and adrenoleukodystrophy (mutations in ABCD1); Metal metabolism disorders such as Wilson's disease (mutation in Wilson's disease protein ATP7B) and hemochromatosis (mutation in human hemochromatosis protein HFE); dysorganic acid metabolism such as methylmalonic acidemia (MUT, MMAA, MMAB, MMADHC) Or mutations in MCEE) and propionic acidemia (mutations in PCCA or PCCB); urea cycle disorders such as ornithine transcarbamylase (OTC) deficiency, arginase (ARG1) deficiency, argininosuccinate lyase (ASL). ) Deficiency, argininosuccinate synthase 1 (ASS1) deficiency, citrin deficiency, carbamoylphosphate synthase 1 (CPSI) deficiency, N-acetylglutamate synthase (NAGS) deficiency and ornithic acid Translocase (ORNT1) deficiency is included.

To treat a subject afflicted with a metabolic disorder, the cloak cells encode a transgene encoding a wild-type form of the gene that is mutated in the subject or an enzyme that is deleted or deleted in the subject (see Table 2). Healthy subject expressing a wild-type form of the gene that is modified in the subject or is deficient in the subject (eg, a subject that does not have a metabolic disorder). The derived cells are one or more of cloaking transgene PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6) (for example, One, two, three, four, five, six, seven or all eight) can be modified to express and administered to a subject with a metabolic disorder. A wild-type form of a gene that is mutated in a subject or a transgene encoding an enzyme that is deleted or deleted in the subject is operably linked to a constitutive promoter, such as CMV or CAG, in cloak cells. It can be constitutively expressed or can be inducibly expressed using one of the inducible expression systems described herein. The wild-type form of the gene that is mutated in the subject or the cloak cells (eg, stem cells) that have been modified to express an enzyme that is deleted or deleted in the subject are administered using methods known to those of skill in the art. Health that expresses a wild-type form of a gene or enzyme that can be differentiated into cells that normally express the gene or enzyme or that can be administered without differentiation, or that is mutated or deleted in the subject. Cells isolated from various subjects, one of cloaking transgene PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6) or Multiples (eg, 1, 2, 3, 4, 5, 5, 6, 7, or all 8) can be modified to express and administered to a subject with a metabolic disorder. If the subject has not yet been diagnosed with the particular mutation prior to treatment, the subject can be evaluated using standard methods to identify mutated genes associated with metabolic disorders, and to which the cloak cells correspond to the wild type. Make sure to express the gene. 800 to 3 billion cloak cells expressing the wild-type form of the gene that has been mutated in the subject (eg, 8×10 ⁸ , 9×10 ⁸ , 1×10 ⁹ , 2×10 ⁹ or 3×10 ^{9 ).} Individual cloak cells) can be injected subcutaneously to produce cloaked subcutaneous tissue producing the corresponding wild-type protein.

Methods of Controlling Cloak Cell Division In one aspect, methods of controlling proliferation of cells at a transplant site in an allogeneic host are provided (eg, to reduce the tumorigenicity of cells at the transplant site or at the transplant site). To reduce the proliferation of cells that have become tumorigenic).

The method provides a cell genetically modified to include at least one mechanism for providing local immunosuppression at the site of transplantation when the cell or population of such cells is transplanted into a cognate host, Genetically modifying a cell division locus (CDL) in a cell, wherein the CDL is one or more loci whose transcript is a dividing cell (eg, one or more immunosuppressive). One or more loci expressed by all dividing cells including the transgene, wherein the genetic modification of the CDL encodes a) a negative selectable marker transcriptionally linked to the DNA sequence encoding the CDL. An inducible exogenous activator (EARC) for the regulation of CDL, including an ablation link (ALIKN) system comprising a DNA sequence Proliferating a genetically modified cell comprising an ALINK system by genetically modifying, comprising one or more of the systems, maintaining the genetically modified cell comprising an ALINK system in the absence of an inducer of a negative selection marker, Or ablating and/or inhibiting the growth of gene-modified cells containing the ALINK system and/or inducing gene-modified cells containing the EARC system by exposing the cells containing the ALINK system to an inducer of a negative selection marker Exposure of an inducer of an activator-based gene expression system to proliferate gene-modified cells containing the EARC system or to induce cells of the EARC system to induce an activator-based gene expression system Maintaining in the absence includes preventing or inhibiting the growth of genetically modified cells, including the EARC system, as well as transplanting the cell or population of cells at the transplant site in a cognate host. Cells modified to control cell division using one or more ALINK and/or EARC systems in one or more CDLs (eg, 2, 3, 4, 4 or more CDLs) May be referred to as "failsafe cells". The number of cells that can be grown from a single fail-safe cell before the cells lose the activity of any system that controls cell division by genetic variation (eg, ALINK or EARC) (eg The number of cell divisions required to "escape" and exhibit uncontrolled cell growth based on mathematical modeling (clone volume) determines the failsafe volume. The failsafe amount depends on the number of ALINKs and the number of ALINK target CDLs. Fail-safe properties are described in more detail in Table 3.

In various embodiments, the CDL is prepared according to Wang et al. , 2015 (as set forth herein, which is incorporated herein by reference in its entirety), identified as an "essential gene". Wang et al. , 2015, are identified by calculating a score (ie, a CRISPR score) for each gene, which score represents the cost of adaptation imposed by gene inactivation. In one embodiment, the CDL has a CRISPR score (CS) of less than about -1.0 (Table 5, column 5).

In various embodiments, the CDL is one or more loci encoding gene products associated with cell division and/or replication (Table 5, column 6). For example, in various embodiments, the CDL is a locus that encodes a gene product associated with one or more of i) cell cycle, ii) DNA replication, iii) RNA transcription and/or protein translation, and iv) metabolism. (Table 5, column 7).

In one embodiment, the CDL is one or more cyclin-dependent kinases involved in regulation of cell cycle progression (eg, control of G1/S G2/M and metaphase to late transition), eg, CDK1, CDK2. , CDK3, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9 and/or CDK11 and the like (Morgan, 2007). In one embodiment, the CDL is one or more cyclins involved in controlling cell cycle progression by activating one or more CDKs, such as cyclin B, cyclin E, cyclin A, cyclin C, cyclin. D, cyclin H, cyclin C, cyclin T, cyclin L, and/or cyclin F (FUN G and POON, 2005). In one embodiment, CDL is one or more loci involved in the late stimulatory complex that regulates the progression of the metaphase to late phase of the M phase of the cell cycle (Peters, 2002). In one embodiment, the CDL is one or more loci involved in centromeric components that control the progression of the metaphase to late phase of the M phase of the cell cycle. In one embodiment, CDL is one or more loci involved in the microtubule component that regulates microtubule motility required for the cell cycle (Cassimeris, 1999).

In various embodiments, the CDL is one or more loci involved in housekeeping. As used herein, the term "housekeeping gene" or "housekeeping locus" refers to one or more genes required to maintain basic cellular function. Housekeeping genes are expressed in all cells of organisms in normal and pathophysiological states.

In various embodiments, the CDL is one or more loci encoding gene products associated with cell division and/or proliferation and has a CRISPR score of less than about -1.0. For example, in one embodiment, the CDL is one or more encoding a gene product associated with one or more of i) cell cycle, ii) DNA replication, iii) RNA transcription and/or protein translation, and iv) metabolism. Multiple loci with a CRISPR score of less than about -1.0. In one embodiment, CDL is also a housekeeping gene.

In some embodiments, the CDL is Cdk1/CDK1, Top2A/TOP2A, Cenpa/CEPNA, Birc5/BIRC5 or Eef2/EEF2. In some embodiments, the CDL is Cdk1/CDK1. In some embodiments, the CDL is Top2A/TOP2A. In some embodiments, the CDL is Eef2/EEF2. In some embodiments, the CDL is Cdk1/CDK1 and Top2A/TOP2A or Cdk1/CDK1 and Eef2/EEF2.

The cell links expression of CDL with expression of a DNA sequence encoding a negative selection marker, thereby allowing drug-induced ablation of mitotically active cells expressing both CDL and a negative selection marker, It can be modified to be a "failsafe" cell. Ablation of proliferating cells is, for example, when cell proliferation is uncontrolled and/or accelerated compared to the normal rate of division of the cells (eg uncontrolled cell division exhibited by cancerous cells) or It may be desirable when the therapeutic need for cells has passed. Ablation of expanded cells links expression of a DNA sequence encoding a negative selectable marker with expression of CDL, resulting in elimination or sufficient inhibition of ALINK-modified expanded cells expressing the CDL locus (contributes to tumorigenesis. Can be achieved by genetic modification to the cells, referred to herein as the "ablation link" (ALINK), which allows inhibition of the cell growth rate to a rate that is too low. Cells expressing the negative selection marker in the presence of a prodrug or other inducer of the negative selection system will either stop growing or die depending on the mechanism of action of the selection marker. The cells can be modified to include homozygous, heterozygous, hemizygous or complex heterozygous ALINK. In one embodiment, negative selectable markers may be introduced at all functional alleles of the CDL to improve ablation fidelity. In one preferred embodiment, a negative selectable marker may be introduced at every functional allele of CDL. The fail-safe system can optionally be used to eliminate all of the cloak cells.

ALINK can be introduced at any position in the CDL, allowing co-expression of the CDL and negative selection marker.

In some embodiments, the ALINK system comprises the herpes simplex virus-thymidine kinase/gancyclovir system, the cytosine deaminase/5-fluorocytosine system, the carboxylesterase/irinotecan system or the iCasp9/AP1903 system.

The DNA encoding the negative selection marker (for example, HSV-TK) includes a host cell whose expression of the negative selection marker expresses the negative selection marker and, inevitably, CDL as an inducer of the negative selection marker (for example, ganciclovir (GCV)). It can be inserted into a CDL (eg, CDK1) in a host cell such that it is killed in the presence of (eg, a prodrug). In this example, the ALINK-modified host cell produces thymidine kinase (TK), and the TK protein converts GCV to GCV monophosphate, which in turn is converted to GCV triphosphate by a cellular kinase. It GCV triphosphate is incorporated into replicating DNA during S phase, leading to DNA elongation and termination of cell apoptosis (Halloran and Fenton, 1998).

The modified HSV-TK gene (Preuss et al., 2010) encodes herein a DNA encoding a negative selectable marker that can be used for ALINK gene modification to selectively ablate cells containing unwanted cell division rates. Is disclosed as an example.

It is contemplated herein that alternative and/or additional negative selection systems may be used with the tools and/or methods described herein. Various negative selectable marker systems are known in the art (eg, dCK.DM (Neschadim et al., 2012)).

For example, various negative selection systems with clinical relevance aim to improve the therapeutic efficacy of conventional cancer therapies with no or minimal side effects, "gene-directed enzyme/prodrugs". Development is ongoing in the field of therapy (GEPT) (Hedley et al., 2007; Nowa et al., 2008). In many cases, GEPT involves using a viral vector to deliver a gene that is absent in a mammalian cell but produces an enzyme in the vicinity of a cancer cell within a cancer cell or a region of a cancer cell, Enzymes can convert relatively non-toxic prodrugs into toxic substances.

HSV-TK/GCV, cytosine deaminase/5-fluorocytosine (CD/5-FC) and carboxylesterase/irinotecan (CE/CPT-11) are negative selectable marker systems that have been evaluated in GEPT preclinical and clinical trials. (Danks et al., 2007; Shah, 2012).

To overcome the potential immunogenicity of the herpes simplex virus type 1 thymidine kinase/gancyclovir (TK/GCV) system, the human deoxycytidine kinase enzyme becomes thymidine active and a non-toxic prodrug: bromovinyl-deoxyuridine (BVdU) A "humanized" suicide system has been developed by engineering this enzyme to act as a negative selection (suicide) system with L, deoxythymidine (LdT) or L-deoxyuridine (LdU) (Neschadim et al. al., 2012).

The CD/5-FC negative selectable marker system is a widely used "suicide gene" system. Cytosine deaminase (CD) is a non-mammalian enzyme obtainable from bacteria or yeasts (eg from Escherichia coli or Saccharomyces cerevisiae, respectively) (Ramnaraine et al., 2003). CD catalyzes the conversion of cytosine to uracil and is a key member of the pyrimidine salvage pathway in protozoa and fungi, but is absent in mammalian cells. 5-Fluorocytosine (5-FC) is an antifungal prodrug that causes low levels of cytotoxicity in humans (Denny, 2003). CD catalyzes the conversion of 5-FC to the genotoxic agent 5-FU and has high levels of toxicity in humans (Ireton et al., 2002).

The CE/CPT-11 system is based on the carboxylesterase enzyme, a serine esterase found in various tissues of mammalian species (Humerickhouse et al., 2000). The anticancer agent CPT-11 is activated by CE to produce an activity called 7-ethyl-10-hydroxycamptothecin (SN-38), a potent mammalian topoisomerase I inhibitor (Wierdl et al. ,, 2001). SN-38 induces accumulation of double-stranded DNA breaks in dividing cells (Kojima et al., 1998).

Another example of a negative selection marker system is the iCasp9/AP1903 suicide system, which is based on modified human caspase-9 fused to human FK506 binding protein (FKBP) and is chemically dimerized with the small molecule AP1903. It allows for somatization, which has been safely tested in humans. Administration of dimerizers induces apoptosis of cells expressing engineered caspase-9 component. This system has several advantages including, for example, low potential immunogenicity, as it is composed of the human gene product, the dimerizing drug expresses the engineered caspase 9 component. Acts on cells only (Strathof et al., 2005). The iCasp/AP1903 suicide system is being tested in the clinical setting (Di Stasi et al., 2011).

It is believed herein that the negative selection marker system of the ALINK system can be replaced by a growth antagonist system. The term “proliferation antagonist” as used herein refers to a natural or engineered compound, the presence of which inhibits (completely or partially) the division of cells. For example, Omomyc ^ER is a fusion protein of a mutant mouse estrogen receptor (ER) domain and a MYC dominant negative Omomyc. When induced with tamoxifen (TAM), the fusion protein Omomyc ^ER localizes to the nucleus, where the dominant negative Omomyc dimerizes with C-Myc, L-Myc and N-Myc, and these Sequester in a complex that is unable to bind to the Myc DNA binding consensus sequence (Soucek et al., 2002). Cells are unable to divide due to lack of Myc activity (Oricchio et al., 2014). Another example of a growth antagonist is A-Fos, a dominant negative for the activating protein 1 (AP1), which inhibits DNA binding in equimolar competition (heterodimer of the oncogenes Fos and Jun) ( Olive et al., 1997). A-Fos also fuses with the ER domain, resulting in its nuclear localization induced by TAM. Omomyc ^ER /tamoxifen or A-Fos ^ER /tamoxifen can be used in place of TK/GCV to become ALINK.

Cells can also be modified to be "failsafe" by operably linking the CDL with an EARC (eg, an inducible activator-based gene expression system). Under these conditions, CDL is expressed only in the presence of the inducer of the inducible activator-based gene expression system, and cells can divide only in that presence. Under these conditions, EARC-modified cells, in the absence of inducer, either arrested or significantly slowed down depending on the mechanism of action and CDL function of the inducible activator-based gene expression system, Or die. The cells can be modified to contain homozygous or complex heterozygous EARC, or modified so that only the EARC modified allele is capable of producing functional CDL. In one embodiment, EARC modifications can be introduced into all alleles of the CDL, for example to provide a mechanism for cell division control.

The EARC can be inserted at any position in the CDL that allows coexpression of the CDL and the activator component of the inducible system in the presence of the inducer.

In one embodiment, "activator" based gene expression systems are preferred over "suppressor" based gene expression systems. For example, when a suppressor is used to suppress CDL, loss of the functional mutation of the suppressor deactivates CDL expression, which allows cell proliferation. In the case of activation-based inhibition of cell division, loss of activator function (mutation) causes CDL expression to cease, which renders cell proliferation impossible.

In some embodiments, the EARC system is a dox-bridge system, a cumate switch inducible system, an ecdysone inducible system, a radio-induced system or a ligand reversible dimerization system.

The dox-crosslinks allow CDL expression in the presence of an inducer (eg, doxycycline or “DOX”) to allow CDL expression, thereby permitting cell division and proliferation (eg, CDL in a host cell). CDK1). A host cell modified with dox-crosslinked EARC may contain the reverse tetracycline trans-activator (rtTA) gene under transcriptional control of a promoter active in dividing cells (eg in CDL) (Urlinger et al., 2000). .. With this targeted insertion, the CDL promoter is no longer available for CDL transcription. To restore CDL transcription, a tetracycline response element promoter (eg, TRE (Agha-Mohammadi et al., 2004)) was inserted in front of the CDL transcript, which resulted in the expression of rtTA and the presence of doxycycline. Will express the CDL gene only in those circumstances. CDL gene expression does not occur in the absence of doxycycline when the only source of CDL expression is the dox-bridge allele. In the absence of CDL expression, EARC-modified cells have their amplification either by cell death, arrest of cell division, or by slowing the cell mitotic rate so far that EARC-modified cells cannot contribute to tumorigenesis. Be disturbed.

As used herein, the term "dox-bridge" refers to the expression of rtTA by an endogenous or exogenous promoter (Gossen et al., 1995), which puts the transcription of the target region under the control of the TRE, thereby targeting transcription. Refers to the mechanism by which promoter activity is separated from a region. As used herein, "rtTA" refers to the reverse tetracycline transactivator element of the tetracycline-inducible system (Gossen et al., 1995), "TRE" is the promoter composed of the TetO operator sequence upstream of the minimal promoter. Refers to. After binding rtTA to the TRE promoter in the presence of doxycycline, transcription of the locus downstream of the TRE promoter is increased. The rtTA sequence may be inserted at the same transcriptional unit as the CDL or at a different position in the genome, as long as the permitting or disallowing status of transcriptional expression of the target region is controlled by doxycycline. dox-crosslinking is an example of EARC.

The introduction of the EARC system into the 5'regulatory region of CDL is also considered herein.

It is contemplated herein that alternative and/or additional inducible activator-based gene expression systems can be used in the tools or methods described herein to generate EARC modifications.

For example, when cell division and/or proliferation is desired, a destabilizing protein domain (Banaszynski et al., 2006) fused to an acting protein product of the encoded CDL is used with a small molecule synthetic ligand to produce a CDL fusion protein. Can be stabilized. In the absence of stabilizers, the destabilized CDL protein will be degraded by the cell, which will then stop growing. When the stabilizing compound is added, it binds to the destabilized CDL protein and the destabilized CDL protein is not degraded, allowing the cells to grow.

For example, transcriptional activator-like effector (TALE) technology (Maeder et al., 2013) can be combined with dimerizer-regulated expression induction (Pollock and Clackson, 2002). TALE technology can be used to create a DNA binding domain designed to be specific for a sequence that is co-located with a minimal promoter that replaces the CDL promoter. The TALE DNA binding domain was also extended by the drug dimerization domain. The latter is capable of binding another engineered protein which has a corresponding dimerization domain and transcriptional activation domain.

For example, a reverse cumate transactivator (rcTA) can be inserted into the 5'untranslated region of CDL so that it is expressed by the endogenous CDL promoter. A 6-repeat (6×CuO) of a cumate operator (Cumate Operator) can be inserted just before the start of transcription of the CDL (ATG). If there is no cumate in the system, rcTA cannot bind to 6xCuO and CDL is not transcribed because 6xCuO is not active. When cumate is added, it forms a complex with rcTA and allows binding to 6xCuO, thus allowing transcription of CDL (Mullick et al., 2006).

For example, by inserting a retinoid X receptor (RXR) and an N-terminal truncated form of the ecdysone receptor (EcR) (VpEcR) fused to the activation domain of Vp16 into the 5'untranslated region of CDL, these It may be co-expressed by the endogenous CDL promoter. The ecdysone responsive element (EcRE) can be inserted into the CDL directly upstream of the start codon with a downstream minimal promoter. The co-expressed RXR and VpEcR can heterodimerize with each other. In the absence of ecdysone or the synthetic drug analog muristerone A, the dimerized RXR/VpEcR cannot bind EcRE and therefore the CDL is not transcribed. In the presence of ecdysone or muristerone A, the dimerized RXR/VpEcR binds EcRE, which causes CDL transcription (No et al., 1996).

For example, transient receptor potential vanilloid-1 (TRPV1) may be inserted into the 5'untranslated region of CDL along with ferritin and co-expressed by the endogenous CDL promoter. Also, a promoter inducible by NFAT (NFATre) may be inserted into the CDL directly upstream of the start codon. Under normal circumstances, the NFAT promoter is not active. However, when exposed to low frequency radio waves, TRPV1 and ferritin form a Ca ⁺⁺ wave that enters the cell, which then converts cytoplasmic-NFAT (NFATc) to nuclear-NFAT (NFATn), which NFATre is actively activated to transcribe CDL (Stanley et al., 2015).

For example, the CDL can be functionally divided into parts/domains: 5'-CDL and 3'CDL to insert the FKBP peptide sequence into each domain. An IRES (internal ribosome entry site) sequence can be placed between the two domains, which are cotranscribed by the CDL promoter but produce two distinct proteins. In the absence of inducer, the two distinct CDL domains are functionally inactive. Upon introduction of a dimerizer such as rapamycin or AP20187, the FKBP peptide dimerizes, resulting in 5'and 3'CDL moieties and reconstituting the active protein (Rollins et al., 2000).

In an embodiment of the method, the genetically modified cells are a set of transgenes, each transgene being a cytoplasmic, membrane-bound or locally acting gene product, the function of which is graft attack. It includes a set of transgenes that encode gene products that either reduce the function of leukocyte and NK cell activation or act as a defense mechanism against leukocyte attack.

Methods for genetically modifying a cell to include at least one mechanism for providing local immunosuppression at the site of transplantation when the cell or population of such cells are transplanted into a cognate host are described, for example, in International Publication No. 2016/141480, the entire teachings of which are incorporated herein by reference.

The set of transgenes comprises one or more of the following genes: PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6). Including. In embodiments, the set of transgene genes comprises PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6).

Optionally, the method comprises expressing in a cell one or more of the following transgenes: TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1 and IFNγR1 d39. Further includes. In embodiments, the TGF-β or biological agent acts locally within the implant environment.

Techniques for introducing various genetic modifications such as transgenes into animal cells are described herein and are generally known in the art.

In an embodiment of the method, the cell is a stem cell, a cell that is subject to genome editing, or a cell that can serve as a source of therapeutic cell types (eg, can be directed to differentiate into lineage restricted or terminally differentiated cells that can be used in cell therapy). Cells or cells of the desired target tissue). In embodiments, the cells are embryonic stem cells, induced pluripotent stem cells, adult stem cells, tissue-specific stem cells, hematopoietic stem cells, mesenchymal stem cells, endothelial stem cells, epithelial stem cells, adipose stem cells or precursor cells, germline stem cells, lung stem cells or Progenitor cells, mammary stem cells, olfactory adult stem cells, hair follicle stem cells, pluripotent stem cells, amniotic stem cells, cord blood stem cells or neural stem cells or progenitor cells. In some embodiments, the cells are derived from a target tissue, such as skin, heart, brain or spinal cord, liver, lung, kidney, pancreas, bladder, bone marrow, spleen, intestine or stomach. In some embodiments, the cells are fibroblasts, epithelial cells or endothelial cells. The cell can be a vertebrate cell, eg, a mammalian cell such as a human or mouse cell.

Techniques for transplanting genetically modified cells into the transplant site of an allogeneic host are described herein and are known in the art.

In various embodiments of any of the disclosed methods, the host has a degenerative disease or condition that can be treated with cell therapy. Examples of such diseases or conditions include, in various embodiments, blindness, arthritis (eg, osteoarthritis or rheumatoid arthritis), ischemia, diabetes (eg, type 1 or type 2 diabetes), multiple sclerosis. Disease, spinal cord injury, stroke, cancer, lung disease, blood disease, Parkinson's disease, Alzheimer's disease, Huntington's disease and neurological diseases such as ALS, enzyme or hormone deficiency, metabolic disorder (eg, lysosomal storage disorder, galactoseemia, maple syrup) Urine, phenylketonuria, glycogen storage disorders, mitochondrial disorders, Friedrich's ataxia, peroxisome disorders, metal metabolism disorders or organic acidemia, autoimmune disorders (eg psoriasis, systemic lupus erythematosus, Grave's disease, inflammatory) Enteropathy, Addison's disease, Sjogren's syndrome, Hashimoto's thyroiditis, vasculitis, autoimmune hepatitis, alopecia areata, autoimmune pancreatitis, Crohn's disease, ulcerative colitis, dermatomyositis), age-related macular degeneration, retinal dystrophy , Infectious diseases, hemophilia, degenerative diseases (eg Charcot-Marie-Tooth disease, chronic obstructive pulmonary disease, chronic traumatic encephalopathy, Creutzfeldt-Jakob disease, cystic fibrosis, cytochrome C oxidase deficiency, Ehlers -Danlos syndrome, essential tremor, progressive ossifying fibrodysplasia, infantile neuroaxonal dystrophy, keratoconus, globular cornea, muscular dystrophy, neuronal ceroid lipofuscinosis, previous disease, progressive supranuclear palsy, Sandhoff Disease, spinal muscular atrophy, retinitis pigmentosa) or age-related disease (eg, atherosclerosis, cardiovascular disease (eg, angina, myocardial infarction), cataract, osteoporosis or hypertension) However, it is not limited to these.

Pharmaceutical Compositions The clonal cells described herein are incorporated into a vehicle for administration to a patient, eg, a human patient undergoing a transplant or suffering from a disease or condition described herein. Can be Pharmaceutical compositions containing cloak cells can be prepared using methods known in the art. For example, such compositions are described, for example, in Physiologically Acceptable Carriers, Excipients or Stabilizers (Remington: The Science and Practice of Pharmacology 22nd edition, Allen, L. Ed. (2013); Incorporated herein) can be used in the desired form (eg, in the form of an aqueous solution).

Cloak cells described herein can be administered in any physiologically compatible carrier, such as a buffered saline solution. Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media. The use of such carriers and diluents is well known in the art. Other examples include liquid media such as Dulbecco's Modified Eagle's Medium (DMEM), sterile saline, sterile phosphate buffered saline, Leibovitz's medium (L15, Invitrogen, Carlsbad, Calif.), dextrose in sterile water and optionally. Other physiologically acceptable liquids are listed. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof as well as in oil. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol such as glycerol, propylene glycol and liquid polyethylene glycol, suitable mixtures thereof and/or vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example sugars or sodium chloride. This solution is preferably sterile and fluid to the extent that easy syringability exists. Preferably, the solution is stable under the conditions of manufacture and storage and is preserved against the contaminating action of microorganisms such as bacteria and fungi, for example through the use of parabens, chlorobutanol, phenol, ascorbic acid, thimerosol and the like. .. The solution of the present invention uses a pharmaceutically acceptable carrier or diluent and, optionally, the other ingredients listed above, followed by filter sterilization and then the cloak cells described herein. Can be prepared by incorporating

For example, a solution containing the pharmaceutical compositions described herein may be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. .. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, sterile aqueous media that can be used are known to those of skill in the art in light of the present disclosure. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations may meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.

Pharmaceutical compositions containing cloak cells in a semi-solid or solid carrier are typically formulated for surgical implantation at the site of implantation or at the site of disease or condition in the subject. It will be appreciated that the liquid composition may also be administered by surgical procedures. In certain embodiments, semi-solid or solid pharmaceutical compositions may include semi-permeable gels, matrices, cell scaffolds, etc., which may be non-biodegradable or biodegradable. For example, in certain embodiments, cloak cells can be sequestered from their surroundings, further allowing the cells to secrete biological molecules (eg, therapeutic agents listed in Table 2) and deliver them to surrounding cells. May be desirable or appropriate.

In other embodiments, different types of degradable gels and networks are utilized for the pharmaceutical compositions of the present invention. For example, degradable materials include biocompatible polymers such as poly(lactic acid), poly(lactic-co-glycolic acid), methyl cellulose, hyaluronic acid, collagen.

In another embodiment, one or more hydrogels are used in the pharmaceutical composition. The one or more hydrogels may include collagen, atelocollagen, fibrin constructs, hydrophilic vinyl and acrylic polymers, polysaccharides such as calcium alginate and poly(ethylene oxide). Further, hydrogels include poly(2-hydroxyethylmethacrylate), poly(acrylic acid), self-assembling peptides (eg RAD16), poly(methacrylic acid), poly(N-vinyl-2-pyrrolidinone), poly(vinyl alcohol). ) And their copolymers with each other and copolymers with hydrophobic monomers such as methylmethacrylate, vinylacetate and the like. Also preferred are hydrophilic polyurethanes containing large poly(ethylene oxide) blocks. Other preferred materials include hydrogels containing an interpenetrating network of polymers, which can be formed by addition or condensation polymerization, the components of which are hydrophilic and hydrophobic monomers such as those listed above. Can be included. In situ forming degradable networks are also suitable for use in the present invention (eg, Anseth, K S et al. J. Controlled Release, 2002; 78:199-209; Wang, D. et al., Biomaterials, 2003; 24:3969-3980; U.S. Patent Application Publication No. 2002/0022676). These in-situ forming materials can be formulated as fluids suitable for injection, and then induced to form hydrogels by various means such as temperature, pH changes and exposure to light in situ or in vivo. .. In one embodiment, the construct contains a fibrin glue containing gel. In another embodiment, the construct contains an atelocollagen-containing gel.

The polymer used to form the matrix can be in the form of a hydrogel. In general, hydrogels are cross-linked polymeric materials that can absorb more than 20% of their weight in water while maintaining a well-defined three-dimensional structure. This definition includes dry cross-linked polymers and water-swellable materials that swell in an aqueous environment. The hydrophilic polymer host, whether the polymer is of biological origin, semi-synthetic or fully synthetic, may be crosslinked to form a hydrogel. Hydrogels can be made from synthetic polymeric materials. Such synthetic polymers represent a reliable source of material that can be tailored for a range of properties and predictable lot-to-lot homogeneity, generally without immunogenicity concerns. The matrix can be a hydrogel formed from self-assembling peptides (eg, US Pat. Nos. 5,670,483 and 5,955,343, US 2002/0160471 and PCT applications). (Discussed in WO 02/062969).

Properties that make hydrogels valuable in drug delivery applications include equilibrium swelling degree, sorption kinetics, solute permeability and their in vivo performance properties. The permeability to the compounds depends in part on the degree of swelling or water content and the rate of biodegradation. It is well within the contemplation of the present invention that the mechanical strength of the gel can be reduced in proportion to the degree of swelling so that the hydrogel can be attached to a substrate so that the composite system enhances the mechanical strength. .. In some embodiments, the hydrogel can be impregnated within a porous substrate to obtain the mechanical strength of the substrate with the useful delivery properties of the hydrogel.

In another embodiment, the pharmaceutical composition comprises a biocompatible matrix made from natural, modified natural or synthetic biodegradable polymers, including homopolymers, copolymers and block polymers and combinations thereof. ..

Examples of suitable biodegradable polymers or polymer classes include fibrin, type I, type II, type III, type IV and V collagen, elastin, gelatin, vitronectin, fibronectin, laminin, thrombin, poly(amino acid), oxidation. Cellulose, tropoelastin, silk, ribonucleic acid, deoxyribonucleic acid; protein, polynucleotide, gum arabic, reconstituted basement membrane matrix, starch, dextran, alginate, hyaluron, chitin, chitosan, agarose, polysaccharide, hyaluronic acid, poly (Lactic acid), poly(glycolic acid), polyethylene glycol, decellularized tissue, self-assembling peptides, polypeptides, glycosaminoglycans, derivatives and mixtures thereof, including but not limited to, discussed within the present disclosure. And any biodegradable polymer. Suitable polymers include poly(lactide) (PLA), polyhydroxybutyrate, polyurethane, polyphosphazene, poly(ethylene glycol)-poly(lactide-co-glycolide, which can be formed from L(+) and D(-) polymers. ) Copolymers, degradable polycyanoacrylates and degradable polyurethanes are also included. For both glycolic acid and lactic acid, intermediate cyclic dimers can be prepared and purified prior to polymerization. These intermediate dimers are called glycolide and lactide, respectively.

Other useful biodegradable polymers or polymer classes include aliphatic polyesters, poly(alkylene oxalates), tyrosine-derived polycarbonates, polyiminocarbonates, polyorthoesters, polyoxaesters, polyamide esters, polyoxa containing amine groups. Ester, poly(propylene fumarate), polyfumarate, polydioxanone, polycarbonate, polyoxalate, poly(α-hydroxy acid), poly(ester), polyurethane, poly(ester urethane), poly(ether urethane), polyanhydride, Included, but not limited to, polyacetate, polycaprolactone, poly(orthoesters), polyamino acids, polyamides and blends and copolymers thereof. Additional useful biodegradable polymers include L- and D-lactic acid stereopolymers, bis(para-carboxyphenoxy)propane and sebacic acid copolymers, sebacic acid copolymers, caprolactone copolymers, poly(lactic acid)/poly( Glycolic acid)/polyethylene glycol copolymer, polyurethane and poly(lactic acid) copolymer, α-amino acid copolymer, α-amino acid and caproic acid copolymer, α-benzyl glutamate and polyethylene glycol copolymer, succinate and poly(glycol) copolymer , Polyphosphazenes, poly(hydroxyalkanoates) and mixtures thereof, but are not limited thereto. Binary and ternary systems are also conceivable.

In general, the material used to form the matrix desirably has (1) mechanical properties suitable for the intended application, and (2) sufficiently intact until the tissue ingrows and heals. (3) does not cause an inflammatory or toxic response, (4) is metabolized in the body after achieving its purpose, (5) easily processed into the desired end product formed, (6) acceptable. It has a good shelf life and (7) is configured to be easily sterilized.

In another embodiment, the population of cloak cells may be administered by use of a scaffold. The composition, shape and porosity of the scaffold can be any of the above. Typically, these three-dimensional biomaterials contain live cells attached to the scaffold, dispersed within the scaffold, or incorporated into the extracellular matrix entrapped in the scaffold. When transplanted into the target area of the body, these grafts become integrated with the host tissue and the transplanted cells are gradually established.

Non-limiting examples of scaffolds that can be used include woven structures such as fabrics, knits, braids, meshes, nonwovens and warp knits; porous foams, semi-porous foams, perforated films or sheets, particulates, beads. And spheres and composite structures that are combinations of the above structures. The non-woven mat is, for example, a fiber composed of a synthetic absorbable copolymer (PGA/PLA) of glycolic acid and lactic acid, which is sold under the trade name of VICRYL suture (Ethicon, Inc., Somerville, NJ). Can be formed using. For example, freeze-drying, as discussed in US Pat. No. 6,355,699, composed of a poly(ε-caprolactone)/poly(glycolic acid) (PCL/PGA) copolymer. Alternatively, a foam formed by a process such as lyophilized can be utilized.

In another embodiment, the framework is felt, which can be composed of multifilament yarns made from bioabsorbable materials. The yarn can be made into a felt using standard textile processing techniques consisting of crimping, cutting, carding and needling. In another embodiment, the cells are seeded on a foam scaffold that can be used as a composite structure.

The framework can be shaped, for example, into a shape useful for filling tissue voids. Thus, the framework can be shaped not only to provide channels for nerve growth, but also to provide support for vascular tissue, muscle tissue, etc. and a scaffold for surrounding tissue. Further, it is understood that the population of cells can be cultured on a preformed non-degradable surgical or implantable device.

The pharmaceutical composition may include a preparation made from cloak cells formulated with a pharmaceutically acceptable carrier or vehicle. Suitable pharmaceutically acceptable carriers include water, salt solutions (such as Ringer's solution), alcohols, oils, gelatins, carbohydrates such as polyvinylpyrrolidine, lactose, amylose or starch, fatty acid esters and hydroxymethylcellulose. Include, but are not limited to, any of those discussed within this disclosure. Such preparations may be sterilized and optionally auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts to influence the osmotic pressure, buffers and colorants. Mixed with. Pharmaceutical carriers suitable for use in the present invention are known in the art and are described, for example, in Pharmaceutical Sciences (17 ^th Ed., Mack Pub. Co., Easton, Pa) and WO 96/05309. Has been done.

Methods of Treatment The cloak cells and compositions described herein can be administered to a subject in need thereof (e.g., a subject that has undergone or has undergone a transplant, or has a disease or condition described herein). Topical administration at or near the site of transplantation, local administration to a site afflicted with a disease or condition (eg, joint injection for treating RA, joint injection for treating wet AMD, central nervous system) (CNS) direct administration (eg, intracerebral, intracerebroventricular, intrathecal, intracisternal or stereotactic administration to treat neurological diseases such as Parkinson's disease), direct to myocardium to treat myocardial infarction Injection, direct injection into myocardium to treat myocardial infarction), intravenous, parenteral, intradermal, transdermal, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intraarterial, intravascular , Inhalation, perfusion, lavage and oral administration. The most suitable route for administration in any given case is the particular cell or composition to be administered, the patient, the method of pharmaceutical formulation, the method of administration (eg, time of administration and route of administration), the age, weight of the patient, It depends on the sex, the severity of the disease to be treated, the patient's diet and the patient's excretion rate. The composition may be administered once or more than once (eg, once a year, twice a year, three times a year, twice a month or once a month). For topical administration, cloak cells can be delivered by any means that places the cell population at the desired location, including implantation with catheters, syringes, shunts, stents, microcatheters, pumps, devices or scaffolds. Can be administered.

As described herein, prior to administration, the cell population may be of the desired cell type (eg, neuron, cardiomyocyte, RPE cell, insulin-producing cell, blood coagulation factor-producing cell, joint fibroblast cell or herein. Can be incubated in the presence or condition of one or more factors that stimulate stem cell differentiation into other cell types described in the text. Such factors are known to those of skill in the art, and will understand that the determination of appropriate conditions for differentiation can be accomplished using routine experimentation. Such factors include growth or trophic factors, chemokines, cytokines, cell products, demethylating agents and, for example, angiogenic, hemangiogenic, angiogenic, skeletal muscle, vascular smooth muscle, pericyte. Other stimuli known to stimulate differentiation of cells, neurons or stem cells along the vascular endothelial pathway or lineage are included. Alternatively, the composition administered to a patient comprises a population of cloak cells having one or more factors that stimulate cell differentiation into a desired cell type, where the cell differentiation is in vivo at a tissue site. Occurs. In some embodiments, cloak cells can be differentiated into organs or tissues in vitro using methods known to those of skill in the art and administered to a subject in need of organ or tissue transplantation.

In some embodiments, cells of a particular cell type are collected from a patient or donor (eg, from an HLA-matched or mismatched donor without, eg, a disease or condition) and one or more (eg, one). 2, 3, 4, 5, 6, 7, or 8) modified to express a cloaking transgene and then administered to a subject. Such an approach is for treating a subject carrying a mutation in a particular gene or for deficient in a particular secreted protein or enzyme, as cloaked donor cells may endogenously express the wild-type version of the gene. Useful for a subject (eg, using cloaked donor cells that endogenously express a protein or enzyme that is deficient in the subject). This approach allows cells in an organ or tissue transplant to have one or more of the cloaking transgenes (eg, 1, 2, 3, 4, 5, 6, 6, 7 before the transplant is performed). One or eight) can be modified so that it can also be used in the treatment of a subject undergoing an organ or tissue transplant.

A subject that can be treated as described herein is a subject that has undergone a transplant or a disease or condition described herein (eg, wet AMD or retinal dystrophy, a neurodegenerative disease (eg Parkinson's disease), Subjects with myocardial infarction, osteoarthritis or RA, diabetes, hemophilia, metabolic disorders or diseases or conditions listed in Table 2). The cells, compositions and methods described herein can be used to treat diseases or conditions caused by or associated with cell loss, protein mutations or defects, or abnormal production of proteins, These can be treated with cell replacement proteins or therapies, production of therapeutic proteins, production of agonist antibodies or production of inhibitory antibodies. The methods described herein can include screening a subject for mutations in genes associated with defective protein production prior to treatment or administration with the compositions described herein. Subjects can be screened for gene mutations using standard methods known to those of skill in the art, such as genetic testing. The methods described herein may also include assessing the symptoms of the disease or condition in the subject prior to treatment or administration with the cloak cells or compositions described herein. The subject can then be evaluated using the same diagnostic test after administration of the cloak cells or composition to determine if the subject's condition has improved. The compositions and methods described herein can be administered as a prophylactic treatment to a patient who has undergone a tissue or organ transplant before the patient exhibits signs of tissue or organ rejection.

The cloaklet cells, compositions and methods described herein are for replacing dead or dying cells in a subject (eg, replacing neurons in a subject suffering from a neurodegenerative disease). Or to replace cardiomyocytes in a subject with myocardial infarction). The plaque cells, compositions and methods described herein can also be used to provide immunosuppression in the area of tissue or organ transplantation or to reduce the risk of tissue or organ transplant rejection. A plaque cell expressing a therapeutic agent such as a protein or agonist antibody, a composition comprising such a cell or a method of administering such a cell is used to mutate or lack a wild-type version of the protein in the subject (eg, , Proteins that are produced, but do not function properly due to genetic mutations, such as truncated proteins or proteins with altered charge, polarity or binding properties; or proteins that are not produced or are produced in inadequate amounts, such as Table 2 Defective protein production associated with the disease or condition of Cloak cells expressing a therapeutic agent (eg, an inhibitory or neutralizing antibody), compositions containing such cells, or methods of administering such cells may be overexpressed proteins or aberrantly produced in a subject. To block or neutralize a protein (eg, a protein produced at a time or location different from the production of that protein in a healthy subject (eg, aberrant protein production associated with the diseases or conditions listed in Table 2)) Can be used for.

Treatment can include administration of various unit doses of cloak cells or compositions containing cloak cells. Each unit dose typically contains a predetermined amount of cloak cells as described herein. The quantity to be administered and the particular route and formulation of administration are within the skill of those in the clinical arts. The unit dose need not be administered as a single infusion but can include continuous infusion over a set period of time. Administration can be performed using catheters, syringes, shunts, stents, microcatheters, pumps, implants with devices or implants with scaffolds. The number of cells administered will vary depending on whether the cells are administered to a tissue, organ or body site associated with the disease or injury or subcutaneously to produce cloaked subcutaneous tissue. You can For administration to a tissue, organ or body part, cloak cells may be, for example, 1×10 ⁴ cells to 1×10 ¹⁰ cells (eg 1×10 ⁴ , 2×10 ⁴ , 3×10 ⁴ cells). ⁴ , 4×10 ⁴ , 5×10 ⁴ , 6×10 ⁴ , 7×10 ⁴ , 8×10 ⁴ , 9×10 ⁴ , 1×10 ⁵ , 2×10 ⁵ , 3×10 ⁵ , 4×10 ⁵ , 5×10 ⁵ , 6×10 ⁵ , 7×10 ⁵ , 8×10 ⁵ , 9×10 ⁵ , 1×10 ⁶ , 2×10 ⁶ , 3×10 ⁶ , 4×10 ⁶ , 5×10 ⁶ , 6×10 ⁶ , 7×10 ⁶ , 8×10 ⁶ , 9×10 ⁶ , 1×10 ⁷ , 2×10 ⁷ , 3×10 ⁷ , 4×10 ⁷ , 5×10 ⁷ , 6×10 ⁷ , 7×10 ⁷ , 8×10 ⁷ , 9×10 ⁷ , 1×10 ⁸ , 2×10 ⁸ , 3×10 ⁸ , 4×10 ⁸ , 5×10 ⁸ , 6×10 ⁸ , 7×10 ⁸ , 8×10 ⁸ , 9×10 ⁸ , 1×10 ⁹ , 2×10 ⁹ , 3×10 ⁹ , 4×10 ⁹ , 5×10 ⁹ , 6×10 ⁹ , 7×10 ⁹ , 8×10 ⁹ , 9×10 ⁹ , 1×10 ¹⁰ cells). The number of cells administered depends on the size of the recipient tissue, organ or body part. For example, 2.5×10 ⁴ to 1×10 ⁵ cells (for example, 2.5×10 ⁴ , 3×10 ⁴ , 4×10 ⁴ , 5×10 ⁴ , 6×10 ⁴ , 7×10 ^4). , 8×10 ⁴ , 9×10 ⁴ or 1×10 ⁵ cells) can be administered (eg, injected) to the subretinal space or specific brain regions of the eye; 1×10 ⁶ to 1×10 ⁸ cells (eg, 1×10 ⁶ , 2×10 ⁶ , 3×10 ⁶ , 4×10 ⁶ , 5×10 ⁶ , 6×10 ⁶ , 7×10 ⁶ , 8×10 ⁶ , 9×10) ⁶ , 1×10 ⁷ , 2×10 ⁷ , 3×10 ⁷ , 4×10 ⁷ , 5×10 ⁷ , 6×10 ⁷ , 7×10 ⁷ , 8×10 ⁷ , 9×10 ⁷ or 1×10 ⁸ cells) can be administered (eg, injected) into the joint, the amount of cells depending on the indirect size; and 5×10 ^{8 to} 5×10 ⁹ cells (eg 5×10 5). ⁽⁸ , 6×10 ⁸ , 7×10 ⁸ , 8×10 ⁸ , 9×10 ⁸ , 1×10 ⁹ , 2×10 ⁹ , 3×10 ⁹ , 4×10 ⁹ or 5×10 ⁹ cells) Can be administered to the myocardium. To produce cloaked subcutaneous tissue, 8×10 ⁸ cells to 3×10 ⁹ cells (eg, 8×10 ⁸ , 9×10 ⁸ , 1×10 ⁹ , 2×10 ⁹ , 3 X10 ⁹ cells) can be administered subcutaneously (eg, injected). Cloak cells may be administered in two or more doses (eg, 2, 3, 4, 5 or more different doses, such as in different sized joints in patients with RA) or in the same dose more than once (eg, 1 It can be administered 2, 3, 4, 5, 6 times or more over time, day, week, month or year). In some embodiments, the cloak cells described herein are administered as a tissue (eg, a tissue that has been propagated and/or differentiated in vitro from cloak cells). In some embodiments, cloak tissue is administered (eg, implanted) with a gel, biocompatible matrix or scaffold.

The compositions described herein prevent or reduce transplant rejection, or ameliorate the symptoms of the diseases or conditions listed in Table 2 (eg, reduce the symptoms of osteoarthritis or RA. For (eg, to reduce inflammation, arthralgia, stiffness, arthralgia, stiffness or immobility); to reduce symptoms of retinal dystrophy or wet AMD (eg, to improve visual acuity, to delay ocular neovascularization) Or to reduce); to reduce symptoms of Parkinson's disease (eg, to reduce tremor, rigidity, bradykinesia or to improve posture or gait); to reduce symptoms of diabetes (eg, insulin To improve the level, to reduce the need for regular insulin infusion); to reduce the symptoms of myocardial infarction (eg, to improve heart function, reduce infarct size); To reduce symptoms (eg, increase levels of blood coagulation factors such as Factor VIII, reduce excessive bleeding, reduce bruise, reduce nosebleeds, reduce joint pain or swelling) Or) sufficient to reduce symptoms of metabolic disorders (eg, to increase appetite, growth or weight gain or to reduce lethargy, weight loss, jaundice, seizures, abdominal pain or vomiting). To be done. Transplant rejection can be assessed using standard methods known to those of skill in the art and is greater than or equal to 5% (eg, 5%, 10%, 15%) compared to the transplant rejection rate typically observed without treatment. , 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more). In some embodiments, administration of the cloak cells or compositions described herein results in a transplant rejection that is comparable to that observed in a subject administered an immunosuppressant drug. Symptoms of the diseases and conditions described herein can be assessed using standard methods known to those of skill in the art and compared to the symptoms prior to administration of the cloak cells or compositions described herein. May be reduced by 5% or more (eg, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) (eg, Subject's condition may improve). These effects include, for example, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks after administration of the compositions described herein. It can occur within a week, 20 weeks, 25 weeks or more. Patients may have 1 month, 2 months, 3 months, 4 months, 5 months, 6 months of administration of cloak cells or compositions depending on the dose and route of administration used for treatment. It can be evaluated later. Depending on the outcome of the assessment, the patient may receive additional treatment.

Combination Therapy In some embodiments, the cloak cells described herein are administered in combination with one or more additional therapeutic agents. The additional therapeutic agent can be administered before administration of the cloak cells, after administration of the cloak cells, or at the same time as administration of the cloak cells. Cloak cells and the additional therapeutic agent can also be administered simultaneously via a co-formulation. Cloak cells and therapeutic agents have overlapping effects of cloak cells and therapeutic agents, and their combined effects are observed in cloak cells or therapeutic agents that have been delivered alone with reduced symptoms or other parameters associated with the disorder. It may also be administered continuously, such that it is larger than one, or larger than that observed in the absence of the others. The effects of cloak cells and therapeutic agents can be partially additive, fully additive, or more than additive (eg, synergistic). Sequential or substantially simultaneous administration of cloak cells and therapeutic agents can be accomplished by any suitable route including, but not limited to, oral, intravenous, intramuscular, topical or subcutaneous routes. .. The cloak cells and the therapeutic agent can be administered by the same or different routes. For example, cloak cells can be administered by subcutaneous injection, while the additional therapeutic agent is administered orally. Cloak cells may be administered immediately before or immediately after the additional therapeutic agent, up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, up to 5 hours, up to 6 hours, up to 7 hours, up to 8 hours, up to 9 hours, up to 10 hours. Up to 11 hours, up to 12 hours, up to 12 hours, up to 13 hours, up to 14 hours, up to 16 hours, up to 17 hours, up to 18 hours, up to 19 hours, up to 20 hours, up to 21 hours, up to 22 hours, up to 23 hours. , Up to 24 hours or 1 to 7, 1 to 14, 1 to 21 or 1 to 30 days before or after.

In one example, the additional therapeutic agent is an immunosuppressive agent commonly given to organ or tissue transplants. Immunosuppressive agents are agents given immediately after transplantation to prevent acute rejection (eg, methylprednisolone, attogum, thymoglobulin, OKT3, basiliximab or daclizumab) or immunosuppressive agents used for maintenance (eg, prednisone, calcineurin). It may be an inhibitor such as cyclosporine, tacrolimus, mycophenolate mofetil, azathioprine or rapamycin. Other immunosuppressive agents given after organ transplantation include corticosteroids (eg methylprednisolone, dexamethasone, prednisolone), cytotoxic immunosuppressants (eg azathioprine, chlorambucil, cyclophosphamide, mercaptopurine, methotrexate), Included are immunosuppressive antibodies (eg, antithymocyte globulin, basiliximab, infliximab), sirolimus derivatives (eg, everolimus, sirolimus) and antiproliferative agents (eg, mycophenolate mofetil, mycophenolate sodium and azathioprine). In this case, the cloak cells are administered at or near the site of transplantation, or the tissue to be transplanted is one or more (e.g. 1, 2, 3, 4, 5, 6, 6, (7 or 8) modified to express a cloaking transgene and the immunosuppressive agent is optionally administered as an additional source of immunosuppression.

For use in treating inflammatory and autoimmune related diseases or conditions, additional agents are disease-modifying anti-rheumatic drugs (DMARDs), biological response modifiers (a type of DMARDs), corticosteroids or non- It may be a steroidal anti-inflammatory drug (NSAID). In some embodiments, the additional agent is prednisone, prednisolone, methylprednisolone, methotrexate, hydroxychloroquine, sulfasalazine, leflunomide, cyclophosphamide, azathioprine or tofacitinib, adalimumab, abatacept, anakinra, quineret, cetolizumab, etaneremab, etaneremab, etaneremab. , Infliximab, rituximab or tocilizumab. In some embodiments, the additional agent is 6-mercaptopurine, 6-thioguanine, abatacept, adalimumab, alemtuzumab (Lemtrada), aminosalicylic acid (5-aminoalicylic acid, sulfasalazine, mesalamine, balsalazide, olsalazine), Antibiotics, antihistamines, anti-TNFα (infliximab, adalimumab, certolizumab pegol, natalizumab), azathioprine, belimumab, β-interferon, calcineurin inhibitors, cetrolizumab, corticosteroids, cromolyn, cyclosporin A, cyclosporin, dimethylfumarate ( Techfidera), etanercept, fingolimod (gilenia), fumarate, glatiramer acetate (copaxone), golimumab, hydroxyurea, IFNγ, IL-11, infliximab, leflunomide, leukotriene receptor antagonist, long-acting β2 agonist, mitoki Santron, mycophenolate mofetil, natalizumab (tysabri), ocrelizumab, pimecrolimus, probiotics (VSL#3), retinoid, rituximab, salicylic acid, short-acting β2 agonist, sulfasalazine, tacrolimus, teriflunomide, theophylline, theophylline. Tocilizumab, ustekinumab (anti-IL-12/IL-23) or vedolizumab (anti-α3β7 integrin). In this case, cloak cells may be administered to replace tissues or organs damaged by inflammatory or autoimmune related diseases or conditions. In another example, the administered cloak cells may be modified to express a biotherapeutic agent (eg, an antibody) directed to the treatment of a particular inflammatory or autoimmune related disease or condition, and Agents may be compounds or common anti-inflammatory agents such as NSAIDs or corticosteroids.

For example, if the disease is rheumatoid arthritis, the additional agent is prednisone, prednisolone and methylprednisolone, methotrexate, hydroxychloroquine, sulfasalazine, leflunomide, cyclophosphamide and azathioprine, tofacitinib, adalimumab, abatacept, anaquinra, quineletumab, certrizumab, It may be one or more of etanercept, golimumab, infliximab, rituximab or tocilizumab. The administered cloak cells can be articular cartilage or bone producing cells. In some embodiments, cloak cells can be modified to produce anti-TNFα antibodies and can be administered in combination with anti-inflammatory agents such as corticosteroids.

In another example, the additional therapeutic agent for use in the treatment of AMD or retinal dystrophy can be an additional biological agent (eg, bevacuzumab, ranibizumab or aflibercept), photodynamic therapy or photocoagulation. .. The administered cloak cells can be retinal cells (eg, RPE cells). In some embodiments, cloak cells can be modified to produce a VEGF inhibitor and administered in combination with photodynamic therapy or photocoagulation.

For use in the treatment of Parkinson's disease, the cloak cells described herein are carbidopa-levodopa, dopamine agonists (eg pramipexole, ropinirole, rotigotine or apomorphine), MAO-B inhibitors (eg selegiline or rasagiline). ), catechol-O-methyl transferase inhibitors (eg entacapone or tolcapone), anticholinergic (eg benztropine or trihexphenidyl), amantadine or deep brain stimulation. The administered cloak cells can be dopaminergic neurons.

Additional agents for treating myocardial infarction include anticoagulants (eg, rivaroxaban, dabigatran, apixaban, heparin, warfarin), antiplatelet agents (eg, aspirin, clopidogrel, dipyramidol, prasugrel, ticagrelor), angiotensin. Converting enzyme inhibitors (eg, benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, trandolapril), angiotensin II receptor blockers (eg, candesartan, eprosartan, irbesartan, losartan, losartan, losartan, losartan, losartan, losartan, losartan, Valsartan), angiotensin receptor neprilysin inhibitor (eg, sucuitril/valsartan), β-blocker (eg, acebuterol, atenolol, betaxolol, bisoprolol, metoprolol, nadolol, propranolol, sotalol), a combination of α and β blockers (eg, Carvedilol, labetalol hydrochloride, calcium channel blockers (eg, amlodipine, diltiazem, felodipine, nifedipine, nimodipine, nisoldipine, verapamil), cholesterol lowering agents (eg, statins (eg, atorvastatin, rosuvastatin), nicotinic acid (eg, lovastatin). ), cholesterol absorption inhibitors (eg ezetimibe/simvastatin), digitalis preparations (eg lanoxin), diuretics (eg amiloride, bumentanide, chlorothiazide, chlorthalidone, furosemide, hydrochlorothiazide, indipamide, spironolactone), vasodilators (Eg isosorbide dinitrate, nesiritide, hydralazine, nitrate, minoxidil), dual antiplatelet therapy (eg aspirin and P2Y12 inhibitors) or cardiac treatment (eg angioplasty, prosthetic heart valve surgery, atherectomy, bypass). Surgery, cardiomyoplasty, heart transplantation, minimally invasive cardiac surgery, radiofrequency ablation, stenting or transmyocardial revascularization) The administered cloak cells may be cardiomyocytes.

For use in treating infections, additional agents are antiviral compounds (eg, vidarabine, acyclovir, ganciclovir, valganciclovir, nucleoside analog reverse transcriptase inhibitors (NRTIs) (eg, AZT (zidovudine), ddI(dld( Didanosine), ddC (zarcitabine), d4T (stavudine) or 3TC (lamivudine)), non-nucleoside reverse transcriptase inhibitor (NNRTI) (eg (nevirapine or delavirdine), protease inhibitor (saquinavir, ritonavir, indinavir or nelfinavir). , Ribavirin or interferon); antibacterial compounds; antifungal compounds; antiparasitic compounds.The administered cloak cells may be immune cells (eg, cells that may facilitate coping with an infectious disease (eg, cloak T cells or B cells)).

For use in the treatment of diabetes, additional agents are insulin, sulfonylureas (eg chlorpropamide, glipizide, glyburide, glimepiride), biguanides (eg metformin), meglitinides (eg repaglinide, nateglinide), thiazolidinediones ( For example, rosiglitazone, pioglitazone), DPP-4 inhibitors (sitagliptin, saxagliptin, linagliptin, alogliptin), SGLT2 inhibitors (eg, canagliflozin, dapagliflozin), α-glucosidase inhibitors (eg, acarbose, miglitol), bile. It may be an acid sequestrant such as colesevelam, aspirin or diet. The administered cloak cells can be pancreatic β cells, which can be optionally modified to express the transgene encoding insulin.

For use in the treatment of hemophilia, additional therapeutic agents include coagulation factors, desmopressin, blood clot preservatives (eg, antifibrinolytic agents such as aprotinin, aminocaproic acid, fibrigogen or tranexamic acid), fibrin. It can be a sealant or physical therapy. The administered cloak cells can be liver sinusoids or endothelial cells that can be optionally modified to express the transgene encoding Factor VIII.

For the treatment of metabolic disorders or diseases, additional therapeutic agents include coenzymes (eg biotin, hydroxycobalamin, riboflavin, pyridoxine, folic acid, thiamine, ubiquinone, tetrahydrobiopterin), bone marrow transplants, organ transplants (eg liver, Kidney or heart transplant), hemodialysis, hemofiltration, exchange transfusion, peritoneal dialysis, medium chain triacylglycerols, miglustat, enzyme replacement therapy or dietary restriction (eg low protein or phenylalanine restriction for subjects with phenylketonuria). Food). Cloak cells are cells that carry a wild-type copy of a gene that is mutated in a subject with a metabolic disorder or cells that endogenously produce an enzyme deficient in a subject with a metabolic disorder (e.g., hepatocytes, kidney cells, heart). Cell or any other cell that carries a wild-type copy of the gene that has been mutated in a subject with a metabolic disorder, or that produces a deficient enzyme in a subject with a metabolic disorder.

For use in the treatment of cancer, additional agents are checkpoint inhibitors, chemotherapeutic agents, biological agents, non-drug therapies (eg radiation therapy, cryotherapy, hyperthermia or surgical resection or tumor tissue). Or it may be an anti-cancer vaccine. Cloak cells can be immune cells (eg, macrophages, natural killer cells, dendritic cells or T cells) that can facilitate coping with cancer.

Checkpoint inhibitors can be divided into at least four major categories: i) agents such as antibodies that directly block the inhibitory pathway on T cells or natural killer (NK) cells (eg nivolumab, pidilizumab/ PD-1 targeting antibodies such as CT-011 and pembrolizumab, antibodies targeting TIM-3 and antibodies targeting LAG-3, 2B4, CD160, A2aR, BTLA, CGEN-15049 or KIR), ii) T cells Or an agent such as an antibody that directly activates the stimulatory pathway on NK cells (eg, an antibody that targets OX40, GITR or 4-1BB), iii) blocks the suppressor pathway on immune cells, or immune cells Agents that deplete the suppressor population of E. coli (eg, CTLA-4 targeting antibodies such as ipilimumab or tremelimumab, antibodies that target VISTA and PD-L2 (eg, PDL2/Ig fusion proteins such as AMP224), Gr1 Or antibodies targeting Ly6G), iv) such as antibodies or small molecules that either directly block the inhibitory pathway on cancer cells or rely on antibody-dependent cellular cytotoxicity to enhance cytotoxicity to cancer cells. Drugs (eg rituximab, antibodies or small molecules targeting PD-L1 (eg MPDL3280A/RG7446; MEDI4736; MSB0010718C; BMS 9365559) and B7-H3 (eg MGA271), B7-H4, Gal-9 or MUC1. Antibodies or small molecule inhibitors that target the). In one embodiment, the checkpoint inhibitor is an inhibitor of HVEM, CD160, CHK1, CHK2, B-7 family ligand or a combination thereof (eg, an inhibitor antibody or a small molecule inhibitor). Such agents described herein are designed and produced, eg, by conventional methods known in the art (eg, Templeton, Gene and Cell Therapy, 2015; Green and Sambrook, Molecular Cloning, 2012). obtain. In one embodiment, the checkpoint inhibitor is an inhibitory antibody (eg, a monospecific antibody such as a monoclonal antibody). The antibody can be, for example, humanized or fully human. In another embodiment, the checkpoint inhibitor is a fusion protein, eg, an Fc receptor fusion protein. In some embodiments, the checkpoint inhibitor is an agent such as an antibody that interacts with the checkpoint protein. In another embodiment, the checkpoint inhibitor is an agent such as an antibody that interacts with a ligand for the checkpoint protein.

Chemotherapeutic agents include alkylating agents, antimetabolites, folic acid analogs, pyrimidine analogs, purine analogs and related inhibitors, vinca alkaloids, epipodopyrlotoxin, antibiotics, L-asparaginase, topoisomerase. Inhibitors, interferons, platinum coordination complexes, anthracenedione-substituted ureas, methylhydrazine derivatives, adrenocortical inhibitors, corticosteroids, progestins, estrogens, antiestrogens, androgens, antiandrogens and gonadotropin releasing hormone analogues are included. Also included are 5-fluorouracil (5-FU), leucovorin (LV), ilenotecan, oxaliplatin, capecitabine, paclitaxel and doxetaxel. Non-limiting examples of chemotherapeutic agents are alkylating agents such as thiotepa and cyclophosphamide; alkyl sulphonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carbocon, metredopa and uredopa; Ethylene amines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylollomelamine; acetogenins (especially bratacin and bratacinone); camptothecin (the synthetic analog topotecan). Bryostatin; calistatin; CC-1065 (including its analogs, calzeresin and bizeresin synthetic analogues); cryptophycin (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (synthetic analogue, KW- 2189 and CB1-TM1); eryuterobin; pancratistatin; sarcodictyin; spongestatin; chlorambucil, chlornafazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide. Nitrogen mustards such as the hydrochloride salt, melphalan, novembichin, phenesterine, predonimustine, trofosfamide, uracil mustard; carmustine, chlorozotocin, phomustine, lomustine, nimustine and ranimustine, such as nitrothaurea antibiotics, eg enediyne antibiotics. , Especially antibiotics such as calicheamicin gammal and calicheamicin omegal; dynemycin including dynemycin A; bisphosphonates such as clodronate; esperamicin; and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophore), Aclacinomycin, Actinomycin, Authoramycin, Azaserine, Bleomycin, Cactinomycin, Carabicin, Caminomycin, Carzinophylline, Chromomycin, Dactinomycin, Daunorubicin, Detorubicin, 6-Diazo-5. -Oxo-L-norleucine, doxorubicin (morpholino-doxorubicin, cyanomorpholino-doki Sorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, mitomycins such as marceromycin, mitomycin C, mycophenolic acid, nogaramycin, olibomycin, peplomycin, porphyromycin, puromycin, puromycin. Keramycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, dinostatin, zorubicin; metabolic antagonists such as metrexate and 5-fluorouracil (5-FU); Folic acid analogs such as trexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyridimine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxyfluridine, enocitabine, phloxuridine. Androgens such as carsterone, dromostanolone, propionate, epithiostanol, mepithiostane, and test lactone; antiadrenal glands such as aminoglutethimide, mitotane, trilostane; folate supplements such as florinic acid; acegraton; aldophosphamide Glycoside; Aminolevulinic acid; Enyluracil; Amsacrine; Vestlabil; Bisanthren; Edatraxate; Defofamin; Demecortin; Diaziquone; Elhomitine; Elliptinium acetate; Epothilone; Etogluside; Gallium nitrate; Hydroxyurea; Lentinan; lonidinin (lon)in Maytansinoids such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidanmol; nitraeline; pentostatin; phenamet; pirarubicin; rosoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; lazoxane; rhizoxin; cizofuran; spirogermanium; 2,3',2''-trichlorotriethylamine; trichothecenes (particularly T-2 toxin, veracrine A, loridin A and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitbronitol; Mitractol; pipobroman; gacitosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids such as paclitaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum distributions such as cisplatin, oxaliplatin and carboplatin. Position complex; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantron; teniposide; edatrexate; daunomycin; aminopterin; xeroda; ibandronate; irinotecan (eg, CPT-11); Topoisomerase inhibitors RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and any of the above pharmaceutically acceptable salts, acids or derivatives. Two or more chemotherapeutic agents can be used in the cocktail administered in combination with the cloak cells described herein. Suitable dosing regimens for combination chemotherapy are known in the art.

Anti-cancer biological agents include cytokines used in the treatment of cancer (eg interferons or interleukins (eg IL-2)). In another embodiment, the biological agent is an anti-angiogenic agent such as an anti-VEGF agent such as bevacizumab. In some embodiments, the biological agent is an immunoglobulin-based biological agent that acts on a target to stimulate an anti-cancer response or antagonizes an antigen important for cancer, such as a monoclonal antibody (e.g., a monoclonal antibody). Humanized antibody, fully human antibody, Fc fusion protein or functional fragment thereof). Such agents include rituximab; daclizumab; basiliximab; palivizumab; infliximab; trastuzumab; gemtuzumab ozogamicin; alemtuzumab; ibritumomabuchiuxetan; adalimumab; omalizumab; tositumomab-Izumab; efalibumab. Natalizumab; Tocilizumab; Panitumab; Ranibizumab; Eculizumab; Certolizumab Pegor; Golimumab; Kanakinumab; Ustekinumab; Ofatumumab; Includes obinutuzumab. Antibody-drug conjugates are also included.

Kits The present invention optionally expresses further one or more of the following transgenes: TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1 and IFNγR1 d39. Cells (eg, the set of cloaking transgenes described herein (eg, PD-L1, H2-M3, Cd47, Cd200, FasL, Ccl21b, Mfge8 and Spi6 one, two A kit comprising (3, 4, 5, 5, 6, 7 or 8) expressing cloak cells) is also featured. In some embodiments, the cloak cells have one or more systems (eg, the ALINK or EARC system) to regulate cell division and/or a therapeutic agent (eg, a transgene encoding a protein or antibody). It is further modified to include. Cloak cells may be provided in a pharmaceutical composition. The kit may further include a syringe for administration of the cloak cells or the pharmaceutical composition and instructions for administering the cloak cells or the pharmaceutical composition for treating the diseases or conditions described herein.

The following examples are provided to further illustrate some embodiments of the invention, but are not intended to limit the scope of the invention, and other procedures, methodologies or techniques known to those of skill in the art. It is understood by their exemplary nature that can be used alternatively.

Example 1: Materials and Methods Construction of a vector expressing a target gene essential for alloresistance A plasmid containing the cDNA sequence of a gene involved in alloresistance was obtained as follows.
PD-L1: Mount Sinai Hospital, clone #V102001
FasL: Mount Sinai Hospital, #75719.
Cd47: Mount Sinai Hospital, #V75535
Cd200: GE Dharmacon, ID# 17470
H2-M3: Mount Sinai Hospital, clone #8188.
Ccl21: Mount Sinai Hospital, clone #V77120.
Mfge8: Mount Sinai Hospital, clone #V72614.
Spi6: Mount Sinai Hospital, clone #V8907

Expression vectors containing these genes of interest (GOI) or luciferase enzymes were created using the Gateway cloning system (Thermo Fisher). The Cd47, Ccl21, Mfge8 and Spi6 cDNAs were obtained in a form containing cDNA flanking attB sites. For H2-M3, Cd200, FasL and PD-L1, primers were designed to amplify the cDNA sequence and an attB site was added (FIG. 15(a)). After PCR amplification, the attB-containing cDNA was recombined into pDONR221 vector (Thermo Fisher, #1256017) by BP (recombination between attB site and attP site) reaction to prepare an entry (pENTRY) clone (FIG. 15 ( b)). The BP reaction involved mixing the attB flanking transgene cDNA with the pDONR221 plasmid in a 1 mL tube along with the buffer and the BP enzyme provided by Invitrogen, where the BP enzyme introduced the GOI into the docking site of the pDONR221 plasmid. Recombine. Insertion of the transgene into the pDONR221 plasmid was confirmed by DNA sequencing (TCAG Sequencing Facility at Center for Applied Genomics, Toronto). Next, the GOENT-containing pENTRY clone was recombined into the target vector via a gateway LR (recombination between attL site and attR site) reaction (FIG. 15(c)). The LR reaction involved mixing the GOI-containing pDONR221 plasmid with the vector of interest in a 1 mL tube with the buffer and the LR enzyme provided by Invitrogen, where the LR enzyme was from pDONR221 to the docking site of the plasmid of interest. Recombine the cassette. The target vector used for all transgene constructions contains the CAGG promoter followed by the gateway entry site, the internal ribosome entry site (IRES) and either the purpuromycin resistance selectable marker or the green fluorescent protein (GFP) reporter. The entire cassette is flanked by transposable PB sites. After LR recombination, the final target vector containing GOI (Fig. 15(d)) was confirmed by restriction enzyme digestion.

ES cell culture, transfection, selection and cloning Mouse ES cells derived from the inbred C57BL/6N mouse strain (Gertsstein 2010) were tested with 15% fetal bovine serum (FBS, tested for compatibility with ES cell culture) and It was cultured in DMEM high glucose supplemented with standard amounts of sodium pyruvate, non-essential amino acids (NEAA), glutamax, penicillin/streptomycin, β-mercapto-ethanol and leukemia inhibitory factor (LIF) (Behringer et al 2014). The cells were cultured on a feeder layer of mitomycin C inactivated mouse embryonic fibroblasts (MEF). Cultures were maintained at 37°C and 5% CO2 in a standard cell culture incubator.

Transfection was performed using JetPRIME reagent (Polyplus, Catalog #14-07) according to the manufacturer's protocol and was performed in 3 steps: 1) Transfection with PD-L1-IRES-GFP target vector only. 2) Transfection with all other transgenes carrying the puromycin selection marker, and 3) transfection with eLuciferase-IRES-GFP transgene.

Step 1: Following transfection with PD-L1-IRES-GFP, cells were plated at low density so that after multiple rounds of expansion after 5-6 days individual cells present as cell aggregates (colonies). Cell clones were selected based on the intensity of GFP expression and then expanded as clonal cell cultures. The clone with the highest and most consistent GFP expression was selected for the next step.

Step 2: 24 hours after transfection with the transgene containing the puromycin selection marker, puromycin was added to the culture medium. On day 3, cells were plated at clonal density, individual colonies were picked and puromycin selection continued until clonally expanded. A large number of these clones were screened in vivo and the clone capable of forming teratomas in an allogeneic setting was designated "NT2".

Step 3: NT2 was transfected with PB-CAG-eLuciferase-IRES-GFP as above and plated at clone density. GFP+ clones were picked and expanded. Ten clones with high levels of GFP expression were selected for further study.

Evaluation of transgene expression levels RNA was isolated from cultures grown on 30 mm culture plates and tumors grown in vivo. Cells were dissociated with trypsin, centrifuged and the supernatant removed. The cell pellet was immediately frozen on dry ice and stored at -80°C. The tumor tissue was dissected, frozen immediately on dry ice, and stored at -80°C. RNA was isolated using the Sigma GeneElute Total RNA Miniprep Kit #RTN350 according to standard protocols. The cDNA was obtained by reverse transcriptase reaction using Qiagen Quantifast Reverse Transcriptase Kit #205313. Quantitative PCR was performed using Sensifast master mix from Bioline, #Bio-98020, gene specific primers and RNA at a 1:50 dilution. Samples were plated in 384-well plates using an Eppendorf epMotion 5070 robot and quantitative PCR was performed on a BioRad CFX384 Real-Time System C1000 thermal cycler according to standard protocols. qPCR data were captured by BioRad CFX Manager 3.1 software and expression levels were calculated using Microsoft Excel.

Teratoma Assay Matrigel Matrix High Concentration (Corning cat# 354248) was diluted 1:1 with cold DMEM medium on ice. 5×10 ⁷ cells were suspended in 500 uL of DMEM and the same amount of Matrigel. 100 ul 5x10 cells of the suspension were injected subcutaneously into each dorsal flank of B6N (syngeneic) or FVB/N (allogeneic). The resulting teratomas formed 2-4 weeks after injection. Teratoma size was measured using calipers and volume was calculated by the formula V=(L×W×H)/2. Tumors grew to approximately 500 mm ² , which is well tolerated and well suited for downstream experiments. All of the transgenes have been delivered to cells containing a “failsafe system” (eg, as described in WO 2016/141480, the entire contents of which are incorporated herein by reference). ). This genetic system allows complete inhibition of cell division with the administration of ganciclovir (GCV). Once the teratoma from the previously described experiment reached 500 mm ² , mice were injected with 50 mg/kg GCV every 2-3 days intraperitoneally for 2-3 weeks. This treatment regimen resulted in an initial brief contraction of the tumor, followed by stabilization of tumor size at 400-500 mm ² 2-3 weeks after treatment. At the end of the experiment, mice were sacrificed and tumors were dissected. A small amount of tissue was snap frozen for RNA extraction while the rest was fixed in 4% paraformaldehyde.

Bioluminescence Imaging Mice that developed teratomas derived from cells transfected with the eLuciferase transgene were injected with 100 μL/25 g body weight (Promega #P104C) of 30 mg/mL VivoGlo Luciferin for 10 minutes before imaging. Animals were anesthetized with isoflurane and placed in an IVIS Lumina II imager (Caliper Life Sciences) driven by Living Image software. The exposure time was set to 5 seconds to 5 minutes depending on the signal intensity.

Histology Fixed tumors were embedded in paraffin, sectioned and stained with hematoxylin/eosin for histological analysis on CMHD pathology cores. Histological images were processed with NDPview2 software.

Example 2: Generation of Cloak Cells A transgene encoding the genes of Table 1 was cloned into an expression vector and the sequences were all known in the art, both by polymerase chain reaction (PCR), restriction enzyme digestion and sequencing. It was verified using the standard method of.

A set of constructs containing the transgenes Cd47, Cd200, FasL and H2-M3 (set 1) were transfected into mouse embryonic stem cells derived from our C57BL/6 mouse ES strain (C2). The presence of the transgene was confirmed by PCR and expression of the expressed protein was recorded by immunohistochemistry (FIGS. 1A-D). A second set of constructs containing the transgene Ccl21, Mfge8, TGF-β and Spi6 (set 2) was transfected into FVB/N-derived ES cells (ES line C2).

Cloak B16F10 melanoma cells were generated using a similar method except that the medium used DMEM containing 10% fetal bovine serum (FBS).

Example 3: Screening process for inhibition of T cell activation A modified in vitro mixed lymphocyte reaction (MLR) assay was used to screen for transgene combinations that yielded the most efficient inhibition of T cell activation. .. Cell lines transfected with the set 1 and set 2 cloaking transgenes from Example 1 were used. Donor OT-I splenocytes were labeled with carboxyfluorescein succinimidyl ester CFSE and 60,000 cells were added to each well of a 96-well plate. ES or melanoma cells were mixed 10:1 with egg expressing cells. 10,000 of these were added to each well of splenocytes. IL-2 was added as a general activator and T cell proliferation was measured 3 days later by flow cytometry (FIGS. 2A-2E). First, cells were gated to contain only CD8+ cells and all conditions were set up in 4 replicates.

The negative control (spleen cells only) resulted in a baseline 6.12% growth rate (Figure 2A). Wild-type B16 melanoma (+10% ovum expressing) cells resulted in a clear acceleration of proliferation to 17.1% (Fig. 2B), whereas cloak cells reduced this proliferation to 9.51% (Fig. 2C). Similar results were obtained for wild type (FIG. 2D) vs. cloaked ES cells (FIG. 2E).

Example 4: Studies with WT and cloak cancer cells in syngeneic and allogeneic B16F10 melanoma cells Since some of the candidate cloaking transgenes are intended to inhibit or regulate the onset of the immune recognition cascade. The effect of the transgene on E. coli is assessed by MLR alone because these events affect the maturation of host APCs and their physical migration into regional lymph nodes, where they subsequently activate naive T and B cells. obtain.

This required an alternative assay that could screen multiple transgene combinations in an in vivo homologous setting. Intraperitoneal and intravenous infusion ES cells with various transgene combinations were tried as options. However, the development of teratomas depends on the aggregation of a minimal number of ES cells (1×10 ^{5 to} 5×10 ⁶ depending on the injection site), making this option incompatible with such screening. However, the mouse melanoma cancer cell line B16F10 derived from C57BL/6 mice is not so limited. Intravenous infusion of less than 5×10 ³ results in the efficient introduction of a wide variety of cancer nodules in the lung. By limiting the number of cells infused, it can be expected that cancer cells will be trapped in the alveoli and form nodules derived from single cells or very few cells. By isolating and genotyping these nodules, the transgene can be identified.

Injection of B16F10 melanoma cells into the bloodstream of C57BL/6 mice (syngeneic graft control) resulted in the formation of cancer nodules in the lung (FIG. 3A, left panel). However, when observed 14 days after injection, small melanoma nodules also formed in the lungs of the negative control (wild type B16F10 melanoma transplanted into allogeneic control FVB mice). However, when melanomas were allowed to grow for 24 days, the nodules almost completely regressed (Fig. 3A, right panel).

The above experiment was repeated by injecting a mixture of cancer cells that expressed a random combination of candidate cloaking genes, created using the PiggyBac transposon system. Pulmonary nodules generated in the allogeneic setting contained the successful combination required to protect the allograft from recognition and rejection (Fig. 3B, right panel). The same immune cloak cells also underwent accelerated development in syngeneic hosts (Fig. 3B, left panel).

Example 5: Noncloak Embryonic Stem Cells Do Not Form Teratomas in Allogeneic Settings As shown in Table 4, wild-type ESCs from C57BL/6 mice are capable of forming teratomas in FVB/N mice. It was confirmed that it was not possible. Similarly, we have also shown that wild-type ESCs derived from the FVB/N background are unable to form teratomas in C57BL/6 hosts. ES cell colonies were dissociated with trypsin, washed once with additive-free DMEM and resuspended in Matrigel HC at a concentration of approximately 50 million cells per milliliter. Recipient mice were anesthetized and 100 microliters were injected subcutaneously in each flank region. Developing teratomas were followed for 12 weeks and confirmed by palpation and caliper volumetric measurements.

Example 6: Cloak ES cells can grow in syngeneic and allogeneic hosts To verify the cloaking ability of candidate transgenes, ESCs are transfected with the same transgene and simultaneously detected by imaging. Luciferase transgene was added. Briefly, ES cells were prepared as described above. The presence of viable cells was repeatedly measured by imaging. The image in FIG. 4 was taken 17 days after injection.

In FIG. 4, the upper panel shows the growth of immune cloak cells in syngeneic hosts, while the lower panel shows the growth of immune cloak cells in allogeneic hosts.

In another experiment, cloaked ES cells from C57BL/6 mice with high expression of eight immunoregulatory transgenes (clone NT2) were treated with different allogeneic mouse strains with mismatched MHC alleles (C3H, FVB/N and Subcutaneous injection into CD1). Red arrows indicate teratomas formed (FIGS. 5A-5C).

Example 7: Mice with Cloak Tissue Do Not Impair Immunity Non-immune cloak (wild type) ESCs are transplanted into mice with preexisting immune cloak tissue and the mice are evaluated to effect the non-immune cloak graft. It was decided whether or not they could be rejected (Fig. 6). The same mouse was imaged several times over 15 days. As shown in the left panel of Figure 6, in syngeneic mouse controls, the grafts were not rejected over time. Among allogeneic FVB mice, the left mouse in the right panel of Figure 6 had an existing immunocloak graft (arrow). The middle mouse in the right panel of Figure 6 was previously transplanted with C57BL/6 allogeneic ESC but rejected the transplant (not bound by theory, the rejection is due to preformed antibodies to C57BL/6 cells). May have). The mouse on the right side of the right panel of Figure 6 was not previously transplanted. All three mice successfully rejected the non-immune cloak graft. The right mouse rejected the graft later, possibly due to the lack of preformed antibodies to C57BL/6 cells.

Similar experiments were performed when wild-type embryonic stem cells were detected up to 9 days after injection into FVB/N mice with cloaked teratoma (FIG. 7). However, on day 12, no evidence of residual cells could be detected. Control animals were C57BL/6 mice also bearing cloaked tumors. The signal in these mice increased over the course of the experiment.

Example 8: Cloaking and Fail-Safe Embryonic Stem Cell Lines The fail-safe C57BL/6 ES cell line (eg, as described in WO/2016/141480) was transformed into five candidate cloaking transgenes (PD-L1, FasL. , Cd47, Cd200 and H2-M3), neither of these transgene strains produced teratomas in the allograft setting. When the set of co-transfected genes was expanded by three additional candidate cloaking genes: Spi6, Ccl21b and Mfge8, 38 clonal lines were generated. One of these lines, NT2, produced teratomas in an allogeneic recipient (FVB). The expression level of the cloaking gene in 38 clonal lines including the NT2 line (see arrows in FIGS. 8A-H) was measured using quantitative PCR (FIGS. 8A-H). Of the 38 clones, NT2 compared to WT ES cells, Ccl21b (16,000x), FasL (25,000x), Cd200 (1700x), Cd47 (16x), Mfge8 (34x), Spi6. (600x) and the highest overexpression of H2-M3 (750x). PD-L1 was not the highest level of expressor among the clones, but 350× expression on ES cells was also a significant increase. The expression of these genes was checked in the Project Grandiose dataset (www.stemformatics.org/project_grandiose) and Ccl21b, FasL, Cd200, PD-L1 and Spi6 expression was below the detection threshold and thus relative to their ES cells. It is also found that the expression is very high. Based on this data, these eight highly activated genes may have a major role in the induction of allograft immune tolerance.

NT2 cells were injected into both C57BL/6 to generate teratomas in the FVB allogeneic setting (FIGS. 11A-11B) and the FVB syngeneic C57B1/6 syngeneic setting. Allogeneic teratomas (n=6) grew steadily from day 12 to day 38. At a size of 500 mm ² , ganciclovir (GCV) treatment was initiated to remove the proliferative components of the tumor (FIGS. 12A-12B, top panel (FIG. 12A) syngeneic teratoma; bottom panel (FIG. 12B) allogeneic teratoma). Tumor). Treatment for 20 days arrested allograft growth. This experiment showed that 1) teratomas derived from failsafe and cloak (NT2) cells responded similarly to GCV treatment, they entered dormancy after brief GCV exposure, 2) teratomas after GCV , Indicates that it remains stable. There are no signs of dormant tissue rejection, and 3) the kinetics of teratoma growth in FVB animals differs from C57BL/6.

High levels of expressed cloaking transgene survive to form teratomas in allogeneic mice. In our system, the cloaking transgene is expressed under the very strong synthetic promoter CAG (shown in the schematic diagram in Figure 19). The CAG promoter is a combination of the cytomegalovirus early enhancer element, the splicer acceptor of the rabbit β-globin gene and the promoter, the first exon and the first intron of the chicken β-actin gene. We performed extensive qPCR analysis for the levels of transgene transcripts in many different ES cell clones, each with different levels of transgene expression. Only ES clones with the highest expression of the cloaking transgene survive in the cognate host.

As shown in FIG. 9, transcript expression levels of immunoregulatory genes associated with cloaking technology varied between ES cell clones. The concentric circles are drawn on a log10 scale. The thick black line is 1x, the next outer ring is 10x, then 100x. The innermost ring is 0.1x. All values are normalized to a positive control, activated leukocytes isolated from mouse lymphoid organs that naturally express the immunomodulatory transgene. The upper left panel shows wild-type ES cells without transgenic modifications for reference, which express little or no associated immunomodulatory transgene. In contrast, clone NT2 and clone 15 (indicated by red squares) both had high expression of the gene and survived in the homologous host. All other clones shown in Figure 9 did not survive in the cognate host.

High expression of the cloaking transgene is also shown in FIG. As shown in FIG. 10, all eight cloaking transgenes in the NT2 cell line and NT2-derived teratomas had expression levels that were in the top 5% of all genes in the ES cell genome, with 5 of the cloaking transgenes. Individuals have expression levels in the top 1% of all genes in the ES cell genome. Expression of these genes is much lower in WT ES cells because only one of these genes has an expression level of the top 5% of all genes in the genome.

Example 9: Cloaked ESCs Contribute to All Three Germ Layers in Allogeneic Teratomas Next, whether immune cloaking allows to expand the full pluripotent developmental potential of ESCs in teratomas. Asked. Teratomas resulting from the injection of cloaked and uncloaked ESCs from C57BL/6 mice into syngeneic and allogeneic hosts were analyzed by histopathology (hematoxylin and eosin staining). 13A-13B (syngeneic host neurons, bone and columnar epithelium in the upper panel (FIG. 13A); and allogeneic host neurons, bone, columnar epithelium and blood vessels in the lower panel (FIG. 13B)) are representatives obtained from both backgrounds. Figure 2 shows a photographic image, demonstrating that expression of the cloaking transgene does not interfere with the normal developmental potential of these ES cells, indicating that the tumor is well vascularized. Both syngeneic and allogeneic tissues showed no immune cell infiltration.

In another experiment, we test whether pluripotent ES cells are able to form cells from all three germ layers (endoderm, ectoderm and mesoderm), thus making them truly pluripotent. It was tested for sex (FIGS. 14A-14D). This was assayed by injecting 10 ⁶ -10 ⁷ cloaked ES cells subcutaneously in mice, allowing them to proliferate and differentiate into tissue masses called teratomas. Teratomas were then removed 3-4 weeks after ES cell injection, tissue sections were cut and stained with H&E. These sections were analyzed under a microscope for cell morphology to determine if all three germ layers were present.

We asked whether the 8 cloaking transgenes, which were inserted into ES cells and expressed at high levels, disrupt their ability to form all three germ layers. They didn't. 14A-14C show three germ layers (ec=ectoderm, shown in FIG. 14A; en=endoderm, shown in FIG. 14C; me=mesoderm, shown in FIG. 14B). FIG. 14D shows blood vessels verifying that these tissues are fully vascularized.

Example 10: ES Cells Expressing Cloaking Transgene Produce a Protein Encoded by the Transgene We used fluorescent antibody-based microscopy to analyze NT2 ES cells (clones with the highest expression. The presence of the protein encoded by the cloaking transgene in 1) was directly confirmed (FIGS. 16A-16H). These data confirm that the protein encoded by the transgene is expressed in ES cells at readily detectable levels expected based on high levels of mRNA expression.

Example 11: ES Cells Expressing High Levels of Cloaking Transgenes Have Typical Morphology and Express Common ES Cell Markers We analyze cloaked ES cells and show that they are It was determined whether the ES cell markers were expressed and retained normal ES cell morphology. Cloaked ES cells have the typical morphology observed in healthy pluripotent ES cells (FIG. 17A) and also stain positive for alkaline phosphatase, a hallmark of healthy pluripotent ES cells. (FIG. 17B). Furthermore, our cloaked ES cells were positive for transcription factors Oct4 (Fig. 18A) and SSEA (Fig. 18B) (both are common markers for normal pluripotent ES cells) using fluorescent antibodies. Stained. These data indicate that ES cells expressing high levels of eight immunoregulatory cloaking transgenes appear as normal ES cells with respect to their morphology and expression of common ES cell markers. Inset shows that staining for Oct4 and SSEA1 (lower left inset) co-localizes with ES cells (visualized using DAPI in upper right inset).

Example 12: IFNγR1 d39 Prevents MHC Upregulation in ES Cells Activated T cells secrete IFNγ, which is expressed on many cell types including ES cell-derived tissues and cells. Bind to the IFNγR1/R2 complex. IFNγ binding to the IFNγ receptor induces upregulation of HLA (MHC in mice) and HLA-related molecules on the cell surface, which increases allograft allograft and immune rejection potential. HLA protein (also called major antigen) differences between donors and recipients are a major cause of rejection in all allografts.

To assess whether disruption of IFNγ signaling inhibits or reduces HLA upregulation, we used the wild-type IFNγR1 or dominant negative IFNγR1 (IFNγR1 d39 lacking 39 amino acids in the cytoplasmic tail) transgene. C57BL/6 ES cells were transfected with the piggyback integrable vector containing These transgenes were expressed under the control of a constitutive CAG promoter upstream of the transgene contained on the same piggyback integration cassette.

Wild type and transfected ES cells were then grown in culture and exposed to 100 ng/mL IFNγ ligand for 24 hours. In wild-type ES and IFNγR1 transfected cells (left and middle panel, respectively, of FIG 20), IFN [gamma] exposure results in increased expression of H-2k ^b and H-2D ^b major histocompatibility surface molecules (MHC class I) However, it was not the case with IFNγR1 d39 cells (right panel of FIG. 20). Exposure to PBS alone had no effect. Expression levels were quantified by detecting MHC class I levels by fluorescent antibody staining and measuring mean fluorescence intensity (MFI) by flow cytometry. These data indicate that IFNγR1 d39 overexpression completely inhibits IFNγ-mediated MHC upregulation in ES cells, which indicates that IFNγR1 d39 expression in ES cells prevents activation of the immune system, It is shown that it can be used to reduce the likelihood of immune rejection. Therefore, IFNγR1 d39 is a useful immunosuppressive transgene that can be expressed by the cloak cells described herein to reduce immune activation and transplant rejection.

Example 13: Administration of Cloak Cells Expressing VEGF Inhibitors to Subjects With Wet AMD According to the methods disclosed herein, physicians of ordinary skill in the art can use wet AMD to treat patients such as human patients. Can be treated to reduce ocular neovascularization or prevent or reduce disease progression. To this end, the physician of ordinary skill in the art would appreciate that PD-L1, under control of a VEGF inhibitor (eg, VEGF-Trap, eg, aflibercept) under the control of a constitutive promoter (eg, CMV or CAG), One or more of HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6) (for example, one, two, three, four, Cloak cells (eg, cloak RPE cells or cloak stem cells differentiated into RPE cells) expressing 5, 6, 7, or 8) can be administered to a human patient. Cloak cells can be administered to a patient to treat wet AMD, for example, by topical administration to the eye (eg, injection into the subretinal space). 25,000 to 100,000 cloak cells (eg, 25,000, 50,000, 75,000 or 100,000 cloak cells) can be administered to each affected eye.

After administration of cloak cells to a patient, a physician of ordinary skill in the art can monitor the expression of VEGF inhibitors and the improvement of the patient in response to treatment by a variety of methods. For example, a physician may use standard approaches to monitor a patient's visual acuity and ocular neovascularization. The finding that the patient's visual acuity improves or does not worsen, or that ocular neovascularization is reduced or does not worsen compared to measurements taken prior to administration of cloak cells, indicates that the patient responds well to treatment. Indicates that Subsequent doses can be determined and administered as needed.

Example 14: Administration of Cloaked Dopaminergic Neurons to a Subject With Parkinson's Disease (PD) According to the methods disclosed herein, physicians of ordinary skill in the art will appreciate the motor symptoms of PD (eg, bradykinesia, bradykinesia, Patients, such as human patients with PD, can be treated to reduce tremor or rigidity) or to prevent or reduce disease progression. To this end, the physician of ordinary skill in the art would under the control of constitutive promoters (eg CMV or CAG) PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21. (Ccl21b), Mfge8 and Serpin B9 (Spi6) one or more (eg 1, 2, 3, 4, 5, 5, 6, 7, or all 8) cloak cells expressing ( For example, dopaminergic neurons modified to express the cloaking transgene or cloak stem cells differentiated into dopaminergic neurons) can be administered to a human patient. Cloak cells can be administered to a patient to treat PD, for example, by local administration to the central nervous system (eg, stereotactic injection into the substantia nigra). 25,000 to 100,000 cloak cells (eg, 25,000, 50,000, 75,000 or 100,000 cloak cells) can be administered. The patient can optionally be administered an additional treatment for PD, such as a dopamine agonist.

After administration of cloak cells to a patient, a physician of ordinary skill in the art can monitor the improvement of the patient in response to treatment by a variety of methods. For example, a physician can monitor a patient's movements using standard neurological tests. The finding that the patient's motor symptoms improve or do not worsen compared to the measurements taken prior to the administration of cloak cells indicates that the patient is responding well to the treatment. Subsequent doses can be determined and administered as needed.

Example 15: Administration of Cloaked Cardiomyocytes to a Subject Affected by Myocardial Infarction According to the methods disclosed herein, a physician of ordinary skill in the art will treat a patient, such as a human patient, who has recently suffered a myocardial infarction. Can improve cardiac function (eg, replace dead or damaged cardiomyocytes). To this end, the physician of ordinary skill in the art would under the control of constitutive promoters (eg CMV or CAG) PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21. (Ccl21b), Mfge8 and Serpin B9 (Spi6) one or more (eg 1, 2, 3, 4, 5, 5, 6, 7, or all 8) cloak cells expressing ( For example, cloaked cardiomyocytes or cloaked stem cells differentiated into cardiomyocytes) can be administered to a human patient. Cloak cells can be administered to a patient to facilitate recovery after myocardial infarction, for example, by local administration to the heart (eg, myocardial infusion). The cells can be injected into the myocardium as monotherapy or the cells can be delivered during bypass surgery or another open heart surgery procedure. 1 to 5 billion cloaked cardiomyocytes (eg, 1×10 ⁶ , 2×10 ⁶ , 3×10 ⁶ , 4×10 ⁶ , 5×10 ⁶ , 6×10 ⁶ , 7×10 ⁶ , 8×10 ⁶ , 9×10 ⁶ , 1×10 ⁷ , 2×10 ⁷ , 3×10 ⁷ , 4×10 ⁷ , 5×10 ⁷ , 6×10 ⁷ , 7×10 ⁷ , 8×10 ⁷ , 9×10 ⁷ or 1×10 ⁸ , 2×10 ⁸ , 3×10 ⁸ , 4×10 ⁸ , 5×10 ⁸ , 6×10 ⁸ , 7×10 ⁸ , 8×10 ⁸ , 9×10 ⁸ , 1×10 ⁹ , 2×10 ⁹ , 3×10 ⁹ , 4×10 ⁹ or 5×10 ⁹ cloak cells) can be administered.

After administration of cloak cells to a patient, a physician of ordinary skill in the art can monitor the improvement of the patient in response to treatment by a variety of methods. For example, a physician may monitor a patient's cardiac function using standard approaches (eg, EKG, echocardiography, angiography, stress testing or nuclear imaging). The finding that a patient's cardiac function is improved or stabilized compared to measurements taken prior to the administration of cloak cells indicates that the patient is responding well to treatment. Subsequent doses can be determined and administered as needed.

Example 16: Administration of Cloak Cells Expressing TNFα Inhibitors to RA Patients According to the methods disclosed herein, a physician of ordinary skill in the art can reduce joint stiffness, swelling or pain by: Patients with rheumatoid arthritis (eg, human patients) can be treated. To this end, the physician of ordinary skill in the art would under the control of constitutive promoters (eg CMV or CAG) PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21. (Ccl21b), one or more of Mfge8 and Serpin B9 (Spi6) (eg 1, 2, 3, 4, 5, 5, 6, 7 or 8 all) and an inducible promoter (eg. , A tetracycline response element) under the control of a TNFα inhibitor (eg, a TNFα inhibitory antibody such as adalimumab) expressing cloak cells (eg, cloaked joint fibroblasts or cloaked joints differentiated into joint fibroblasts) Fibroblasts) can be administered to human patients. Cloak cells can be administered to a patient to treat RA, for example, by topical administration to a joint (eg, injection into an arthritic joint such as a hand joint). 1 to 100 million cloak joint fibroblasts expressing anti-inflammatory biological substances (eg 1×10 ⁶ , 2×10 ⁶ , 3×10 ⁶ , 4×10 ⁶ , 5×). 10 ⁶ , 6×10 ⁶ , 7×10 ⁶ , 8×10 ⁶ , 9×10 ⁶ , 1×10 ⁷ , 2×10 ⁷ , 3×10 ⁷ , 4×10 ⁷ , 5×10 ⁷ , 6× 10 ⁷ , 7×10 ⁷ , 8×10 ⁷ , 9×10 ⁷ or 1×10 ⁸ cloak indirect fibroblasts) can be administered to each affected joint. If the patient experiences a recurrence of RA symptoms, the patient can be treated with tetracycline or doxycycline to drive the expression of the TNFα inhibitor. Tetracycline or doxycycline can be discontinued when the patient's relapse has resolved.

After administration of cloak cells and tetracycline or doxycycline to a patient, a physician of ordinary skill in the art can monitor TNFα inhibitor expression and the patient's improvement in response to therapy by a variety of methods. For example, a physician may monitor pain, swelling and stiffness of a patient's joints using standard approaches. The finding that the patient's joint pain, swelling or stiffness is reduced compared to the measurements obtained prior to administration of cloak cells indicates that the patient is responding well to the treatment. Subsequent doses can be determined and administered as needed.

Example 17: Administration of Insulin-expressing Cloak Cells to a Type 1 Diabetic Subject According to the methods disclosed herein, a physician of ordinary skill in the art would identify patients (eg, human patients) with Type 1 diabetes. Treatment can increase insulin levels. To this end, the physician of ordinary skill in the art would appreciate that PD-L1, HLA-G under the control of constitutive promoters (eg CMV or CAG) and insulin under the control of constitutive promoters (eg CMV or CAG). (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6), one or more (e.g., one, two, three, four, five, Administering to a human patient cloaklet cells (eg, cloaked stem cells, cloaked pancreatic β-cells or cloaked stem cells differentiated into pancreatic β-cells) expressing 6, 7, or 8 all) it can. Cloak cells can be administered to a patient to treat type 1 diabetes, eg, by subcutaneous injection (eg, to create cloaked subcutaneous tissue). Insulin-expressing 1 to 3 billion cloak cells (eg, 1×10 ⁶ , 2×10 ⁶ , 3×10 ⁶ , 4×10 ⁶ , 5×10 ⁶ , 6×10 ⁶ , 7×10 ^{6 ).} , 8×10 ⁶ , 9×10 ⁶ , 1×10 ⁷ , 2×10 ⁷ , 3×10 ⁷ , 4×10 ⁷ , 5×10 ⁷ , 6×10 ⁷ , 7×10 ⁷ , 8×10 ^7. , 9×10 ⁷ or 1×10 ⁸ , 2×10 ⁸ , 3×10 ⁸ , 4×10 ⁸ , 5×10 ⁸ , 6×10 ⁸ , 7×10 ⁸ , 8×10 ⁸ , 9×10 ⁸ 1×10 ⁹ , 2×10 ⁹ or 3×10 ⁹ cloak cells) can be administered subcutaneously.

After administration of cloak cells to a patient, a physician of ordinary skill in the art can monitor the onset of improvement in the patient in response to treatment by a variety of methods. For example, a physician can use standard approaches to monitor insulin levels or symptoms of type 1 diabetes (eg, unintended weight loss, fatigue or blurred vision). The finding that a patient's insulin levels are increasing or that the symptoms of type 1 diabetes are reduced compared to measurements taken prior to administration of cloak cells indicates that the patient is responding well to treatment. Indicates. Subsequent doses can be determined and administered as needed.

Example 18: Administration of Cloak Cells Expressing Factor VIII to Hemophilia Patients According to the methods disclosed herein, a physician of ordinary skill in the art will understand that patients with hemophilia (eg, humans). The patient) may be treated to increase levels of blood coagulation factors or to reduce excessive bleeding or bruising. To this end, the physician of ordinary skill in the art would appreciate that PD-L1, HLA under the control of constitutive promoters (eg CMV or CAG) and factor VIII under the control of constitutive promoters (eg CMV or CAG). One or more of G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6) (e.g. 1, 2, 3, 4, 5) One, six, seven, or all eight) cloaked cells (eg, cloak stem cells, cloak endothelial cells or cloak stem cells differentiated into endothelial cells) can be administered to a human patient. Cloak cells can be administered to a patient to treat hemophilia, eg, by subcutaneous infusion (eg, to make cloaked subcutaneous tissue). 1 million to 3 billion cloak cells expressing Factor VIII (eg, 1×10 ⁶ , 2×10 ⁶ , 3×10 ⁶ , 4×10 ⁶ , 5×10 ⁶ , 6×10 ⁶ , 7×10 ⁶ , 8×10 ⁶ , 9×10 ⁶ , 1×10 ⁷ , 2×10 ⁷ , 3×10 ⁷ , 4×10 ⁷ , 5×10 ⁷ , 6×10 ⁷ , 7×10 ⁷ , 8×10 ⁷ , 9×10 ⁷ or 1×10 ⁸ , 2×10 ⁸ , 3×10 ⁸ , 4×10 ⁸ , 5×10 ⁸ , 6×10 ⁸ , 7×10 ⁸ , 8×10 ⁸ , 9×10 ⁸ , 1×10 ⁹ , 2×10 ⁹ or 3×10 ⁹ cloak cells) can be administered subcutaneously.

After administration of cloak cells to a patient, a physician of ordinary skill in the art can monitor the onset of improvement in the patient in response to treatment by a variety of methods. For example, a physician may use standard approaches to monitor Factor VIII levels or symptoms of hemophilia (eg, excessive bleeding or frequent bruising). The finding that a patient's Factor VIII levels are increased or the symptoms of hemophilia are decreased compared to measurements taken prior to administration of cloak cells indicates that the patient responds well to treatment. Indicates. Subsequent doses can be determined and administered as needed.

Example 19: Administration of Cloak Cells Expressing Glucocerebrosidase to a Gaucher's Disease Subject According to the methods disclosed herein, a physician of skill in the art can reduce Glucocerebroside accumulation, or Gaucher's disease. A patient, such as a human patient having Gaucher disease, may be treated to reduce the symptoms of (eg, fatigue, anemia, low platelet count, hypertrophy of the liver or spleen). To this end, the physician of ordinary skill in the art would appreciate that PD-L1, HLA under the control of constitutive promoters (eg CMV or CAG) and glucocerebrosidase under the control of constitutive promoters (eg CMV or CAG). One or more of G(H2-M3), Cd47, Cd200, FASLG(FasL), Ccl21(Ccl21b), Mfge8 and Serpin B9(Spi6) (e.g. 1, 2, 3, 4, 5; One, six, seven or all eight) cloak cells (eg cloak stem cells) can be administered to a human patient. Cloak cells can be administered to a patient by, for example, subcutaneous injection (eg, to create cloaked subcutaneous tissue) to treat Gaucher disease. 1 to 3 billion cloak cells expressing glucocerebrosidase (eg, 1×10 ⁶ , 2×10 ⁶ , 3×10 ⁶ , 4×10 ⁶ , 5×10 ⁶ , 6×10 ⁶ , 7×). 10 ⁶ , 8×10 ⁶ , 9×10 ⁶ , 1×10 ⁷ , 2×10 ⁷ , 3×10 ⁷ , 4×10 ⁷ , 5×10 ⁷ , 6×10 ⁷ , 7×10 ⁷ , 8× 10 ⁷ , 9×10 ⁷ or 1×10 ⁸ , 2×10 ⁸ , 3×10 ⁸ , 4×10 ⁸ , 5×10 ⁸ , 6×10 ⁸ , 7×10 ⁸ , 8×10 ⁸ , 9× 10 ⁸ , 1×10 ⁹ , 2×10 ⁹ or 3×10 ⁹ cloak cells) can be administered subcutaneously.

After administration of cloak cells to a patient, a physician of ordinary skill in the art can monitor the onset of improvement in the patient in response to treatment by a variety of methods. For example, a physician may use standard approaches to monitor glucocerebrosid accumulation or symptoms of Gaucher disease (eg, fatigue, anemia, low platelet count, liver or spleen hypertrophy). The finding that the patient has a reduced accumulation of glucocerebrosides or symptoms of Gaucher disease compared to measurements taken prior to administration of cloak cells indicates that the patient is responding well to treatment. Subsequent doses can be determined and administered as needed.

Example 20: Administration of Cloak Cells to a Subject Receiving a Liver Transplant According to the methods disclosed herein, a physician of ordinary skill in the art may undergo a liver transplant to reduce the risk of transplant rejection. Existing patients (eg, human patients) can be treated. To this end, the physician of ordinary skill in the art will appreciate that PD-L1, HLA-G (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 under the control of constitutive promoters (eg CMV or CAG). (Ccl21b), Mfge8 and Serpin B9 (Spi6) one or more (eg one, two, three, four, five, six, seven or all eight) cloak cells ( For example, cloak stem cells, cloak hepatocytes or cloak stem cells differentiated into hepatocytes) can be administered to a human patient. Cloak cells can be administered to a patient to reduce the risk of transplant rejection, for example, by injection near the liver or a site in the transplanted liver. 1 million to 100 billion cloak cells (eg 1×10 ⁶ , 2×10 ⁶ , 3×10 ⁶ , 4×10 ⁶ , 5×10 ⁶ , 6×10 ⁶ , 7×10 ⁶ , 8×10). ⁶ , 9×10 ⁶ , 1×10 ⁷ , 2×10 ⁷ , 3×10 ⁷ , 4×10 ⁷ , 5×10 ⁷ , 6×10 ⁷ , 7×10 ⁷ , 8×10 ⁷ , 9×10 ⁷ , 1×10 ⁸ , 2×10 ⁸ , 3×10 ⁸ , 4×10 ⁸ , 5×10 ⁸ , 6×10 ⁸ , 7×10 ⁸ , 8×10 ⁸ , 9×10 ⁸ , 1×10 ⁹ , 2×10 ⁹ , 3×10 ⁹ , 4×10 ⁹ , 5×10 ⁹ , 6×10 ⁹ , 7×10 ⁹ , 8×10 ⁹ , 9×10 ⁹ , 1×10 ¹⁰ , 2×10 ¹⁰ 3, 3×10 ¹⁰ , 4×10 ¹⁰ , 5×10 ¹⁰ , 6×10 ¹⁰ , 7×10 ¹⁰ , 8×10 ¹⁰ , 9×10 ¹⁰ or 1×10 ¹¹ cloak cells) or its liver Can be administered nearby.

After administration of cloak cells to a patient, a physician of ordinary skill in the art can monitor the onset of improvement in the patient in response to treatment by a variety of methods. For example, a physician may use standard approaches to monitor patients for symptoms that predict transplant rejection. Findings of the results of transplant rejection comparable to those observed in immunosuppressant-treated subjects indicate that the patient responds well to treatment. Subsequent doses can be determined and administered as needed.

Example 21: Administration of Closque and Fail-Safe Cells Expressing Insulin to Type 1 Diabetic Subjects According to the methods disclosed herein, physicians of ordinary skill in the art will understand that patients with type 1 diabetes (eg, Human patients) can be treated to increase insulin levels. To this end, the physician of ordinary skill in the art would appreciate that PD-L1, HLA-G under the control of constitutive promoters (eg CMV or CAG) and insulin under the control of constitutive promoters (eg CMV or CAG). (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6), one or more (e.g., one, two, three, four, five, Administering to a human patient cloaklet cells (eg, cloaked stem cells, cloaked pancreatic β-cells or cloaked stem cells differentiated into pancreatic β-cells) expressing 6, 7, or 8 all) it can. Cloak cells can also be modified to allow their growth control by linking the expression of CDL with the expression of a DNA sequence encoding a negative selection marker. For example, cloak cells can be modified to contain homozygous ALINKS (eg, HSV-TK system) at two CDL loci (eg, Cdk1 and Top2A). Cloak cells can be administered to a patient to treat type 1 diabetes, eg, by subcutaneous injection (eg, to create cloaked subcutaneous tissue). Insulin-expressing 1 to 3 billion cloak cells (eg, 1×10 ⁶ , 2×10 ⁶ , 3×10 ⁶ , 4×10 ⁶ , 5×10 ⁶ , 6×10 ⁶ , 7×10 ^{6 ).} , 8×10 ⁶ , 9×10 ⁶ , 1×10 ⁷ , 2×10 ⁷ , 3×10 ⁷ , 4×10 ⁷ , 5×10 ⁷ , 6×10 ⁷ , 7×10 ⁷ , 8×10 ^7. , 9×10 ⁷ or 1×10 ⁸ , 2×10 ⁸ , 3×10 ⁸ , 4×10 ⁸ , 5×10 ⁸ , 6×10 ⁸ , 7×10 ⁸ , 8×10 ⁸ , 9×10 ⁸ 1×10 ⁹ , 2×10 ⁹ or 3×10 ⁹ cloak cells) can be administered subcutaneously.

After administration of cloak cells to a patient, a physician of ordinary skill in the art can monitor the onset of improvement in the patient in response to treatment by a variety of methods. For example, a physician can use standard approaches to monitor insulin levels or symptoms of type 1 diabetes (eg, unintended weight loss, fatigue or blurred vision). The finding that a patient's insulin levels are increasing or that the symptoms of type 1 diabetes are reduced compared to measurements taken prior to administration of cloak cells indicates that the patient is responding well to treatment. Indicates. Subsequent doses can be determined and administered as needed.

A physician of ordinary skill in the art can also monitor the size of cloaked subcutaneous tissue. If the cloaked subcutaneous tissue appears to be becoming tumorigenic, the physician can administer ganciclovir to the subject to remove proliferating cloak cells. Nonproliferative cloak cells do not express CDL and are therefore not removed by ganciclovir treatment.

Example 22: Administration of Insulin-expressing Cloak and Fail-Safe Cells to a Type 1 Diabetic Subject According to the methods disclosed herein, a physician of ordinary skill in the art would understand that patients with type 1 diabetes (eg, Human patients) can be treated to increase insulin levels. To this end, the physician of ordinary skill in the art would appreciate that PD-L1, HLA-G under the control of constitutive promoters (eg CMV or CAG) and insulin under the control of constitutive promoters (eg CMV or CAG). (H2-M3), Cd47, Cd200, FASLG (FasL), Ccl21 (Ccl21b), Mfge8 and Serpin B9 (Spi6), one or more (e.g., one, two, three, four, five, Administering to a human patient cloaklet cells (eg, cloaked stem cells, cloaked pancreatic β-cells or cloaked stem cells differentiated into pancreatic β-cells) expressing 6, 7, or 8 all) it can. Cloak cells can also be modified to allow their growth control by linking the expression of CDL with the expression of a DNA sequence encoding an inducible activator system. For example, dox-crosslinks are inserted into two CDLs (eg, Cdk1 and Top2A) so that in the presence of an inducer (eg, doxycycline) dox-crosslinks allow CDL expression, thereby promoting cell division and proliferation. To allow, homozygous modifications can be generated in both CDL in cloak cells. Cloak cells can be administered to a patient to treat type 1 diabetes, eg, by subcutaneous injection (eg, to create cloaked subcutaneous tissue). Insulin-expressing 1 to 3 billion cloak cells (eg, 1×10 ⁶ , 2×10 ⁶ , 3×10 ⁶ , 4×10 ⁶ , 5×10 ⁶ , 6×10 ⁶ , 7×10 ^{6 ).} , 8×10 ⁶ , 9×10 ⁶ , 1×10 ⁷ , 2×10 ⁷ , 3×10 ⁷ , 4×10 ⁷ , 5×10 ⁷ , 6×10 ⁷ , 7×10 ⁷ , 8×10 ^7. , 9×10 ⁷ or 1×10 ⁸ , 2×10 ⁸ , 3×10 ⁸ , 4×10 ⁸ , 5×10 ⁸ , 6×10 ⁸ , 7×10 ⁸ , 8×10 ⁸ , 9×10 ⁸ 1×10 ⁹ , 2×10 ⁹ or 3×10 ⁹ cloak cells) can be administered subcutaneously.

After administration of cloak cells to a patient, a physician of ordinary skill in the art can monitor the onset of improvement in the patient in response to treatment by a variety of methods. For example, a physician can use standard approaches to monitor insulin levels or symptoms of type 1 diabetes (eg, unintended weight loss, fatigue or blurred vision). The finding that a patient's insulin levels are increasing or that the symptoms of type 1 diabetes are reduced compared to measurements taken prior to administration of cloak cells indicates that the patient is responding well to treatment. Indicates. Subsequent doses can be determined and administered as needed. If the doctor determines that the subject requires higher levels of insulin, the doctor can expand the cloak cells by treating the subject with doxycycline. Once the desired level of insulin is reached, treatment with doxycycline can be stopped and cloak cell growth is stopped.

While this disclosure has been described with reference to particular specific embodiments, various modifications thereof will be apparent to those skilled in the art. All examples provided herein are included solely for purposes of illustrating the present disclosure and are not intended to limit the present disclosure in any way. Any drawings provided herein are intended only to illustrate various aspects of the present disclosure and are not intended to scale or limit the present disclosure in any way. The claims appended hereto should not be limited to the preferred embodiments described in the above description, but should be given their broadest interpretation consistent with the specification as a whole. All prior art disclosures listed herein are incorporated herein by reference in their entirety.

Claims (192)

A cell genetically modified to include at least one mechanism for providing local immunosuppression at the site of transplantation when transplanted into an allogeneic host,
-A set of transgenes, each transgene being a gene product that is cytoplasmic, membrane bound or locally acting, the function of which is:
a) mitigating the activation and function of antigen presenting cells,
b) mitigating graft attack leukocyte activity or cytolytic function,
c) alleviating the macrophage cytolytic function and phagocytosis of allograft cells,
d) Inducing apoptosis in graft-attacking leukocytes,
e) including a set of transgenes that encode a gene product that is one or more of mitigating local inflammatory proteins and f) protecting against leukocyte-mediated apoptosis, the set of transgenes comprising: Gene: PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8 and Serpin B9 or Spi6 or PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG. Alternatively, a cell containing two or more genes encoding a biological substance that acts as an agonist of FasL, Ccl21 or Ccl21b, Mfge8, or Serpin B9 or Spi6. The set of transgenes comprises the following genes: PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8 and Serpin B9 or Spi6 or PD-L1, HLA-G or. H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8 or 3, 4, 5, 6, 7 of genes encoding biological substances acting as agonists of Serpin B9 or Spi6 Or the cell of claim 1, comprising all eight. The set of transgene genes is PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8 and Serpin B9 or Spi6 or PD-L1, HLA-G or H2-M3. , Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8 and a gene encoding a biological substance that acts as an agonist of Serpin B9 or Spi6. Following the transgene: TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1 and IFNganmaaru1 d39 or TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, The cell according to any one of claims 1 to 3, further comprising one or more genes encoding a biological substance that acts as an agonist of Dad1 or IFNγR1 d39. The cell of claim 4, wherein the TGF-β or biological agent acts locally within the graft environment. 6. A cell according to any one of claims 1 to 5, which is a source of stem cells, cells according to genome editing and/or therapeutic cell types. Embryonic stem cell, pluripotent stem cell, induced pluripotent stem cell (iPSC), hematopoietic stem cell, mesenchymal stem cell, endothelial stem cell, epithelial stem cell, adipose stem cell or progenitor cell, reproductive stem cell, lung stem cell or progenitor cell, mammary gland stem cell, olfactory adult Stem cells, hair follicle stem cells, intestinal stem cells or progenitor cells, pluripotent stem cells, amniotic stem cells, cord blood stem cells, neural stem cells or progenitor cells, adult stem cells, somatic stem cells, tissue-specific stem cells, totipotent stem cells, fibroblasts, Monocyte progenitor cells, B cells, exocrine cells, pancreatic progenitor cells, endocrine progenitor cells, hepatoblasts, myoblasts, preadipocytes, hepatocytes, chondrocytes, smooth muscle cells, K562 human erythroid leukemia cell line, bone Cells, synovial cells, tendon cells, ligament cells, meniscus cells, adipocytes, dendritic cells, natural killer cells, skeletal muscle cells, cardiomyocytes, erythroid-megakaryocytic cells, eosinophils, macrophages, T cells , Islet β cells, neurons, cardiomyocytes, blood cells, exocrine precursor cells, duct cells, acinar cells, α cells, β cells, δ cells, PP cells, cholangiocytes, white or brown adipocytes, hormone secreting cells, epidermis Keratinocytes, epithelial cells, kidney cells, germ cells, skeletal joint synovial cells, periosteal cells, perichondrocytes, chondrocytes, endothelial cells, pericardial cells, meningeal cells, keratinocyte precursor cells, keratinocyte stem cells, pericytes , Glial cells, ependymal cells, cells isolated from amnion or placental membrane, serosa cells, somatic cells or skin, heart, brain or spinal cord, liver, lung, kidney, pancreas, bladder, bone marrow, spleen, intestine or stomach The cell according to claim 6, which is derived from the cell. Further comprising at least one mechanism for controlling cell proliferation,
a) a genetic modification of one or more cell division loci (CDL), said CDL being one or more endogenous or exogenous loci, the transcripts of which are expressed by dividing cells. One or more endogenous or exogenous loci, wherein the genetic modification is
i) an ablation link (ALINK) system comprising a DNA sequence encoding a negative selectable marker transcriptionally linked to the DNA sequence encoding the CDL, and ii) an inducible activator operably linked to the CDL 8. A cell according to any one of claims 1 to 7, which comprises a genetic modification that is one or more of the extrinsic activator of regulation of CDL (EARC) system, including a based gene expression system. The genetic modification of the CDL is
a) a DNA vector containing the above-mentioned LINK system,
b) performing a targeted substitution of the CDL with one or more of a DNA vector comprising the EARC system, and c) one or more of the ALINK system and a DNA vector comprising the EARC system, wherein the ALINK and/or 9. The cell of claim 8, wherein each EARC system is operably linked to said CDL. The genetic modification of the CDL containing the ALINK system is homozygous, heterozygous, hemizygous or complex heterozygous, and/or the genetic modification of the CDL containing the EARC system is 10. The cell according to claim 8 or 9, which causes activation of the CDL only by an inducer of a gene expression system based on a sex activator. The cell according to any one of claims 8 to 10, wherein the CDL is one or more of the loci listed in Table 5. 12. The cell of claim 11, wherein the CDL encodes a gene product that functions in one or more of cell cycle, DNA replication, RNA transcription, protein translation and metabolism. 13. The CDL of claim 12, wherein the CDL is one or more of Cdk1/CDK1, Top2A/TOP2A, Cenpa/CENPA, Birc5/BIRC5 and Eef2/EEF2, preferably the CDL is Cdk1 or CDK1. cell. The ALINK system includes herpes simplex virus-thymidine kinase/gancyclovir system, cytosine deaminase/5-fluorocytosine system, carboxylesterase/irinotecan system or iCasp9/AP1903 system, preferably, the ALINK system is herpes simplex virus-thymidine system. The cell according to any one of claims 8 to 13, which is a kinase/gancyclovir system. The EARC system is a dox-crosslinking system, a cumate switch inducing system, an ecdysone inducing system, a radiowave inducing system or a ligand reversible dimerization system, preferably the EARC system is a dox-crosslinking system. Item 14. The cell according to any one of items 8 to 13. One or more of PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8 and Serpin B9 or Spi6 is above the expression level of the corresponding endogenous gene in activated leukocytes. The cell according to any one of claims 1 to 15, which is expressed at the level of. All eight of PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8 and Serpin B9 or Spi6 are above the expression level of the corresponding endogenous gene in activated leukocytes. The cell of claim 16, which is expressed at a level. The cell according to any one of claims 1 to 17, wherein the PD-L1 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12. 19. The HLA-G or H2-M3 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:16 or SEQ ID NO:15. Cells. 20. The cell of any one of claims 1-19, wherein the Cd47 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4. 21. The cell of any of claims 1-20, wherein the CD200 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6. 22. The cell according to any one of claims 1 to 21, wherein the FASLG or FasL transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO:9. 23. The cell of any one of claims 1-22, wherein the Ccl21 or Ccl21b transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:1. 24. The cell of any one of claims 1-23, wherein the Mfge8 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:13 or SEQ ID NO:14. 25. The cell of any one of claims 1-24, wherein the Serpin B9 or Spi6 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:7. The cell according to any one of claims 4 to 25, wherein the IFNγR1 d39 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:17. 27. The cell of any one of claims 1-26, wherein the two or more transgenes are operably linked to a constitutive promoter. The constitutive promoter is a CAG promoter, cytomegalovirus (CMV) promoter, EF1α promoter, PGK promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, HSV tk promoter, mouse mammary tumor virus (MMTV). 28. The cell according to claim 27, which is selected from the group consisting of a promoter, an HIV LTR promoter, a Moloney virus promoter, an Epstein Barr Virus (EBV) promoter and a Rous sarcoma virus (RSV) promoter. 29. The cell of any of claims 1-28, further comprising a transgene encoding a therapeutic agent. 30. The cell of claim 29, wherein the therapeutic agent is a protein or antibody. The cell according to claim 30, wherein the antibody is an inhibitory antibody or an agonist antibody. 32. The therapeutic agent is a drug listed in Table 2 or a wild type version of a gene known to be mutated in cancer, enzyme or hormone deficiency, metabolic disease or degenerative disease. The cell according to any one of claims. The therapeutic agent is an inducible expression selected from the group consisting of a tetracycline response element, a light induction system, a radioactive gene system, a cumate switch induction system, an ecdysone induction system, a destabilization domain system or a ligand reversible dimerization system. 33. The cell according to any one of claims 29 to 32, which is expressed using the system. The therapeutic agent is a CAG promoter, cytomegalovirus (CMV) promoter, EF1α promoter, PGK promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter. The LTR promoter of HIV, the promoter of Moloney virus, the Epstein-Barr virus (EBV) promoter, and the Rous sarcoma virus (RSV) promoter, expressed using a constitutive promoter. The cell according to any one of claims. A population of genetically modified cells comprising the cells of any of claims 1-34. A method for providing local immunosuppression at a transplant site in a homologous host, wherein the population of cells according to any one of claims 1 to 34 or the genetically modified cells according to claim 35 is provided in a homologous host. A method comprising transplanting at a transplant site. A method for providing local immunosuppression at a transplant site in a cognate host, comprising:
(I) providing a cell, and (ii) expressing a set of transgenes in said cell, each transgene being a cytoplasmic, membrane-bound or locally acting gene product. , Its function is
a) mitigating the activation and function of antigen presenting cells,
b) mitigating graft attack leukocyte activity or cytolytic function,
c) alleviating the macrophage cytolytic function and phagocytosis of allograft cells,
d) Inducing apoptosis in graft-attacking leukocytes,
e) alleviating local inflammatory proteins, and f) encoding and expressing a gene product that is one or more of protecting against leukocyte-mediated apoptosis, the set of transgenes comprising: : PD-L1, HLAG or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8 and Serpin B9 or Spi6 or PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, A method comprising two or more genes encoding biological substances that act as agonists of Ccl21 or Ccl21b, Mfge8 or Serpin B9 or Spi6. The set of transgenes comprises the following genes: PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8 and Serpin B9 or Spi6 or PD-L1, HLA-G or. H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8 or 3, 4, 5, 6, 7 of genes encoding biological substances acting as agonists of Serpin B9 or Spi6 38. The method of claim 37, or including all eight. The set of transgene genes is PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8 and Serpin B9 or Spi6 or PD-L1, HLA-G or H2-M3. 38. The method of claim 37, which comprises a gene encoding a biological agent that acts as an agonist of Cd47, Cd200, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8 and Serpin B9 or Spi6. Following the transgene: TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1 and IFNganmaaru1 d39 or TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, 40. The method of any one of claims 37-39, further comprising expressing one or more genes encoding biological agents that act as agonists of Dad1 or IFNγR1 d39. 41. The method of claim 40, wherein the TGF-[beta] or biological agent acts locally within the implant environment. 42. The method of any one of claims 37-41, wherein the cells are a source of stem cells, cells that are subject to genome editing and/or therapeutic cell types. The cells are embryonic stem cells, induced pluripotent stem cells, induced pluripotent stem cells (iPSC), hematopoietic stem cells, mesenchymal stem cells, endothelial stem cells, epithelial stem cells, adipose stem cells or progenitor cells, reproductive stem cells, lung stem cells or progenitor cells, Mammary gland stem cells, olfactory adult stem cells, hair follicle stem cells, intestinal stem cells or progenitor cells, multipotent stem cells, amniotic stem cells, cord blood stem cells, neural stem cells or progenitor cells, adult stem cells, somatic stem cells, tissue-specific stem cells, totipotent stem cells , Fibroblast, monocyte progenitor cell, B cell, exocrine cell, pancreatic progenitor cell, endocrine progenitor cell, hepatoblast, myoblast, preadipocyte, hepatocyte, chondrocyte, smooth muscle cell, K562 human erythroid system Leukemia cell line, bone cell, synovial cell, tendon cell, ligament cell, meniscus cell, adipocyte, dendritic cell, natural killer cell, skeletal muscle cell, cardiomyocyte, erythroid-megakaryocytic cell, eosinophil , Macrophages, T cells, islet β cells, neurons, cardiomyocytes, blood cells, exocrine precursor cells, duct cells, acinar cells, α cells, β cells, δ cells, PP cells, cholangiocytes, white or brown adipocytes, Hormone-secreting cells, epidermal keratinocytes, epithelial cells, renal cells, germ cells, skeletal joint synovial cells, periosteal cells, perichondrocytes, chondrocytes, endothelial cells, pericardial cells, meningeal cells, keratinocyte precursor cells, keratinocytes Stem cells, pericytes, glial cells, ependymal cells, cells isolated from amnion or placental membrane, serosa cells, somatic cells or skin, heart, brain or spinal cord, liver, lung, kidney, pancreas, bladder, bone marrow, spleen 43. The method of claim 42, which is a cell derived from the intestine or the stomach. A method for controlling the growth of cells according to any one of claims 1 to 7 at a transplant site in a homologous host,
a) genetically modifying a cell division locus (CDL) in a cell, said CDL being one or more endogenous or exogenous loci, the transcripts of which are expressed by dividing cells. One or more endogenous or exogenous loci, wherein the genetic modification of the CDL is
i) an ablation link (ALINK) system comprising a DNA sequence encoding a negative selectable marker transcriptionally linked to the DNA sequence encoding the CDL, and ii) an inducible activator operably linked to the CDL Genetic modification comprising one or more of the inducible exogenous activator of regulation of CDL (EARC) system, including a gene expression system based on
b) transplanting said cell or population of said cells at a transplant site in a homologous host, and c) maintaining said genetically modified cells comprising said ALINK system in the absence of an inducer of said negative selection marker. By allowing the growth of said genetically modified cell comprising said ALINK system and/or exposing said cell comprising said ALINK system to said inducer of said negative selection marker, said genetic modification comprising said ALINK system. Ablating or inhibiting cell proliferation, and/or ii) comprising the EARC system by exposing the genetically modified cell comprising the EARC system to an inducer of a gene expression system based on the inducible activator By allowing the growth of said genetically modified cells or maintaining said cells containing said EARC system in the absence of said inducer of said inducible activator-based gene expression system, said EARC system is A method comprising preventing or inhibiting the growth of said genetically modified cell comprising. 45. The method of any of claims 36-44, wherein the homologous host is a mammal. 46. The method of claim 45, wherein the allogeneic host is a mouse. 46. The method of claim 45, wherein the allogeneic host is a human. 48. The method of any of claims 36-47, wherein the host has a degenerative disease or condition that can be treated with cell therapy. The disease or condition is blindness, arthritis, ischemia, diabetes, multiple sclerosis, spinal cord injury, stroke, cancer, lung disease, blood disease, Parkinson's disease, Alzheimer's disease, Huntington's disease, ALS, enzyme or hormone deficiency, 49. The method of claim 48, which is a metabolic disease, autoimmune disease, age-related macular degeneration, retinal dystrophy, infection, hemophilia, myocardial infarction, degenerative disease or age-related disease. One or more of PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8 and Serpin B9 or Spi6 is above the expression level of the corresponding endogenous gene in activated leukocytes. 50. The method of any of claims 37-49, which is expressed at the level of. All eight of PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8 and Serpin B9 or Spi6 are above the expression level of the corresponding endogenous gene in activated leukocytes. 51. The method of claim 50, which is expressed at a level. 52. The method of any of claims 37-51, wherein the PD-L1 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:11 or SEQ ID NO:12. 53. The HLA-G or H2-M3 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:16 or SEQ ID NO:15. the method of. 54. The method of any one of claims 37-53, wherein the Cd47 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4. 55. The method of any one of claims 37-54, wherein the CD200 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6. 56. The method of any one of claims 37-55, wherein the FASLG or FasL transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO:9. 57. The method of any one of claims 37-56, wherein the Ccl21 or Ccl21b transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:1. 58. The method of any one of claims 37-57, wherein the Mfge8 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:13 or SEQ ID NO:14. 59. The method of any one of claims 37-58, wherein the Serpin B9 or Spi6 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:9. 60. The method of any of claims 40-59, wherein the IFNγR1 d39 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:17. 61. The method of any one of claims 37-60, wherein the one or more transgenes are operably linked to a constitutive promoter. The constitutive promoter is a CAG promoter, cytomegalovirus (CMV) promoter, EF1α promoter, PGK promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, HSV tk promoter, mouse mammary tumor virus (MMTV). 62. The method of claim 61, selected from the group consisting of a promoter, an HIV LTR promoter, a Moloney virus promoter, an Epstein Barr Virus (EBV) promoter and a Rous sarcoma virus (RSV) promoter. 63. The method of any one of claims 37-62, wherein the cell further comprises a transgene encoding a therapeutic agent. 64. The method of claim 63, wherein the therapeutic agent is a protein or antibody. 65. The method of claim 64, wherein the antibody is an inhibitory antibody or an agonist antibody. 66. The method of any one of claims 63-65, wherein the therapeutic agent is a drug listed in Table 2 or a wild-type version of a gene that is mutated in the subject. The therapeutic agent is an inducible expression selected from the group consisting of a tetracycline response element, a light induction system, a radioactive gene system, a cumate switch induction system, an ecdysone induction system, a destabilization domain system or a ligand reversible dimerization system. 67. The method of any of claims 63-66, which is expressed using the system. The therapeutic agent is a CAG promoter, cytomegalovirus (CMV) promoter, EF1α promoter, PGK promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter. 67. The LTR promoter of HIV, the promoter of Moloney virus, the Epstein-Barr virus (EBV) promoter and the Rous sarcoma virus (RSV) promoter expressed using a constitutive promoter selected from the group consisting of: 63-66. The method according to any one of claims. A composition comprising the cells according to any one of claims 1 to 34. 70. The composition of claim 69, further comprising a pharmaceutically acceptable excipient. A kit comprising the cells according to any one of claims 1 to 34 or the composition according to claim 69 or 70. 37. A method of performing treatment of a disease or condition in a subject in need thereof, wherein the cell of any one of claims 1-34 or the composition of claim 69 or 70 is administered to said subject. A method including: The disease or condition is blindness, arthritis, ischemia, diabetes, multiple sclerosis, spinal cord injury, stroke, cancer, lung disease, blood disease, Parkinson's disease, Alzheimer's disease, Huntington's disease, ALS, enzyme or hormone deficiency, 73. A metabolic disease, autoimmune disease, age-related macular degeneration, retinal dystrophy, infection, hemophilia, myocardial infarction, degenerative disease, age-related disease or a disease or condition listed in Table 2, according to claim 72. The method described. 74. The method of claim 72 or 73, wherein the cells are differentiated into a lineage restricted cell type prior to administration to the subject. 77. The method of claim 74, wherein the disease or condition is myocardial infarction and the cells are differentiated into cardiomyocytes. 75. The method of claim 74, wherein the disease or condition is blind and the cells are differentiated into photoreceptor cells. 75. The method of claim 74, wherein the disease or condition is spinal cord injury, Parkinson's disease, Huntington's disease or Alzheimer's disease, and the cells are differentiated into neurons. 75. The method of claim 74, wherein the disease or condition is multiple sclerosis, and the cells are differentiated into glial cells. 79. The method of any one of claims 72-78, wherein the cells are administered locally to a tissue or body site in need of the cells or the therapeutic agent. 80. The method of any of claims 72-79, wherein the cells are administered intravenously, subcutaneously, intramuscularly, transdermally, intradermally, parenterally, intraarterially, intravascularly or by perfusion. 81. The method of claim 80, wherein the cells are administered by subcutaneous injection to produce cloaked subcutaneous tissue. 80. The method of any one of claims 72-79, wherein the cells are administered as tissue. 83. The method of claim 82, wherein the tissue is administered with a gel, biocompatible matrix or cell scaffold. 80. The method of any one of claims 72-79, wherein the cells are administered in an amount of 25,000 to 5,000,000 cells. 84. The method of any one of claims 80-83, wherein the cells are administered in an amount of 800,000,000 to 3,000,000 cells. 86. The method of any one of claims 72-85, further comprising administering an additional therapeutic agent. 87. The method of claim 86, wherein the additional therapeutic agent is administered prior to administration of the cells. 87. The method of claim 86, wherein the additional therapeutic agent is administered after administration of the cells. 87. The method of claim 86, wherein the additional therapeutic agent is administered concurrently with the administration of the cells. The additional therapeutic agents include immunosuppressants, disease-modifying anti-rheumatic drugs (DMARDs), biological response modifiers (a type of DMARDs), corticosteroids or nonsteroidal anti-inflammatory drugs (NSAIDs), prednisone, prednisolone, Methylprednisolone, methotrexate, hydroxychloroquine, sulfasalazine, leflunomide, cyclophosphamide, azathioprine, tofacitinib, adalimumab, abatacept, anakinra, kinelet, certolizumab, etanercept, golimumab, infliximab, rituximab, 6-turizimab, tocilitumab, tocilicizumab, toricitumab, tocilicizumab, toricusimab, tocilitumab, tocilicizumab, toricusimab, or tociritumab, tocilicizumab, toricatimab, tocilicizumab, rituximab, or tociritumab Abatacept, adalimumab, alemtuzumab, aminosalicylate, antibiotics, antihistamine, anti-TNFα, azathioprine, belimumab, β-interferon, calcineurin inhibitor, certolizumab, corticosteroids, cromolyn, cyclosporin A, cyclosporine, dimethyl fumarate, etanermodin, etanermocept, , Fumarate, glatiramer acetate, golimumab, hydroxyurea, IFNγ, IL-11, leflunomide, leukotriene receptor antagonist, long-acting β2 agonist, mitoxantrone, mycophenolate mofetil, natalizumab, ocrelizumab, pimecrolimus, Probiotics, retinoids, salicylic acid, short-acting β2 agonists, sulfasalazine, tacrolimus, teriflunomide, theophylline, tocilizumab, ustekinumab or vedolizumab, bevacucizumab, ranibizumab or aflibercept), photodynamic therapy, photocoagulation, carbidopa-levodave Dopamine agonist, MAO-B inhibitor, catechol-O-methyltransferase inhibitor, anticholinergic, amantadine, deep brain stimulant, anticoagulant, antiplatelet agent, angiotensin converting enzyme inhibitor, angiotensin II receptor blocker , Angiotensin receptor neprilysin inhibitor, β blocker, combined α and β blocker, calcium channel blocker, cholesterol lowering drug, nicotinic acid, cholesterol absorption inhibitor, digitalis preparation, diuretic, vasodilator, antiplatelet agent Combination therapy, cardiac treatment, antiviral compound, nucleoside analog reverse transcriptase inhibitor (NRTI), non-nucleoside reverse transcriptase inhibitor (NNRTI), protease inhibitor, antibacterial compound, antifungal compound , Antiparasitic compound, insulin, sulfonylurea, biguanide, meglitinide, thiazolidinedione, DPP-4 inhibitor, SGLT2 inhibitor, α-glucosidase inhibitor, bile acid sequestrant, aspirin, diet, coagulation factor, desmopressin, blood clot Preservative, fibrin sealant, physiotherapy, coenzyme, bone marrow transplant, organ transplant, hemodialysis, hemofiltration, exchange transfusion, peritoneal dialysis, medium chain triacylglycerol, miglustat, enzyme replacement therapy, checkpoint inhibitor, chemotherapy 90. The method of any one of claims 86-89, which is a drug, biological agent, radiation therapy, cryotherapy, hyperthermia, surgical resection or tumor tissue or anti-cancer vaccine. 91. The method of any of claims 72-90, further comprising controlling proliferation of the cells. The cell comprises an ALINK system, and the method of controlling proliferation comprises
i) maintaining the cells containing the ALINK system in the absence of an inducer of the negative selectable marker to allow growth of the cells containing the ALINK system, or ii) containing the ALINK system 92. The method of claim 91, comprising ablating or inhibiting proliferation of the cells, including the ALINK system, by exposing the cells to the inducer of the negative selectable marker. The cell comprises an EARC system, and the method of controlling proliferation comprises
i) exposing the cells containing the EARC system to an inducer of a gene expression system based on the inducible activator to allow growth of the cells containing the EARC system, or ii) the EARC Maintaining or preventing the growth of said cells comprising said EARC system by maintaining said cells comprising said system in the absence of said inducer of said inducible activator-based gene expression system, Item 91. The method according to Item 91. 94. The method of any of claims 72-93, wherein the cells are removed after completion of the treatment. 73. A cell according to any one of claims 1-34 or a composition according to claim 69 or 70 for use in treating a disease or condition in a subject in need thereof. The disease or condition is blindness, arthritis, ischemia, diabetes, multiple sclerosis, spinal cord injury, stroke, cancer, lung disease, blood disease, Parkinson's disease, Alzheimer's disease, Huntington's disease, ALS, enzyme or hormone deficiency, The metabolic disease, autoimmune disease, age-related macular degeneration, retinal dystrophy, infectious disease, hemophilia, myocardial infarction, degenerative disease, age-related disease or a disease or condition listed in Table 2, according to claim 95. The described cell. 73. A cell according to any one of claims 1 to 34 or a composition according to claim 69 or 70 for use in providing local immunosuppression to a transplant site in an allogeneic host. Following the transgene: TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1 and IFNganmaaru1 d39 or TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, The cell of claim 1, further comprising one or more genes encoding biological agents that act as agonists of Dad1 or IFNγR1 d39. 99. The cell of claim 98, wherein the TGF-β or biological agent acts locally within the implant environment. 2. The cell of claim 1, which is a source of stem cells, cells that are subject to genome editing and/or therapeutic cell types. Embryonic stem cell, pluripotent stem cell, induced pluripotent stem cell (iPSC), hematopoietic stem cell, mesenchymal stem cell, endothelial stem cell, epithelial stem cell, adipose stem cell or progenitor cell, reproductive stem cell, lung stem cell or progenitor cell, mammary gland stem cell, olfactory adult Stem cells, hair follicle stem cells, intestinal stem cells or progenitor cells, pluripotent stem cells, amniotic stem cells, cord blood stem cells, neural stem cells or progenitor cells, adult stem cells, somatic stem cells, tissue-specific stem cells, totipotent stem cells, fibroblasts, Monocyte progenitor cells, B cells, exocrine cells, pancreatic progenitor cells, endocrine progenitor cells, hepatoblasts, myoblasts, preadipocytes, hepatocytes, chondrocytes, smooth muscle cells, K562 human erythroid leukemia cell line, bone Cells, synovial cells, tendon cells, ligament cells, meniscus cells, adipocytes, dendritic cells, natural killer cells, skeletal muscle cells, cardiomyocytes, erythroid-megakaryocytic cells, eosinophils, macrophages, T cells , Islet β cells, neurons, cardiomyocytes, blood cells, exocrine precursor cells, duct cells, acinar cells, α cells, β cells, δ cells, PP cells, cholangiocytes, white or brown adipocytes, hormone secreting cells, epidermis Keratinocytes, epithelial cells, kidney cells, germ cells, skeletal joint synovial cells, periosteal cells, perichondrocytes, chondrocytes, endothelial cells, pericardial cells, meningeal cells, keratinocyte precursor cells, keratinocyte stem cells, pericytes , Glial cells, ependymal cells, cells isolated from amnion or placental membrane, serosa cells, somatic cells or skin, heart, brain or spinal cord, liver, lung, kidney, pancreas, bladder, bone marrow, spleen, intestine or stomach 101. The cell of claim 100, which is a derived cell. Further comprising at least one mechanism for controlling cell proliferation,
a) a genetic modification of one or more cell division loci (CDL), said CDL being one or more endogenous or exogenous loci, the transcripts of which are expressed by dividing cells. One or more endogenous or exogenous loci, wherein the genetic modification is
i) an ablation link (ALINK) system comprising a DNA sequence encoding a negative selectable marker transcriptionally linked to the DNA sequence encoding the CDL, and ii) an inducible activator operably linked to the CDL 2. The cell of claim 1, comprising a genetic modification that is one or more of the extrinsic activator (EARC) system of regulation of CDL, including a gene expression system based on it. The genetic modification of the CDL is
a) a DNA vector containing the above-mentioned LINK system,
b) performing a targeted substitution of the CDL with one or more of the EARC system-containing DNA vector and c) one of the ALINK system and the EARC system-containing DNA vector, wherein the ALINK and/or 103. The cell of claim 102, wherein each EARC system is operably linked to said CDL. The genetic modification of the CDL containing the ALINK system is homozygous, heterozygous, hemizygous or complex heterozygous, and/or the genetic modification of the CDL containing the EARC system is 103. The cell of claim 102, which results in activation of said CDL only by an inducer of a sex activator-based gene expression system. 103. The cell of claim 102, wherein the CDL is one or more of the loci listed in Table 5. 106. The cell of claim 105, wherein the CDL encodes a gene product that functions in one or more of cell cycle, DNA replication, RNA transcription, protein translation and metabolism. 107. The CDL of claim 106, wherein the CDL is one or more of Cdk1/CDK1, Top2A/TOP2A, Cenpa/CENPA, Birc5/BIRC5 and Eef2/EEF2, preferably the CDL is Cdk1 or CDK1. cell. The ALINK system includes herpes simplex virus-thymidine kinase/gancyclovir system, cytosine deaminase/5-fluorocytosine system, carboxylesterase/irinotecan system or iCasp9/AP1903 system, preferably, the ALINK system is herpes simplex virus-thymidine system. 103. The cell of claim 102, which is a kinase/gancyclovir system. The EARC system is a dox-crosslinking system, a cumate switch inducing system, an ecdysone inducing system, a radiowave inducing system or a ligand reversible dimerization system, preferably the EARC system is a dox-crosslinking system. Item 102. The cell according to Item 102. One or more of PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8 and Serpin B9 or Spi6 is above the expression level of the corresponding endogenous gene in activated leukocytes. The cell of claim 1, which is expressed at the level of. All eight of PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8 and Serpin B9 or Spi6 are above the expression level of the corresponding endogenous gene in activated leukocytes. 112. The cell of claim 110, which is expressed at a level. The cell of claim 1, wherein the PD-L1 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:11 or SEQ ID NO:12. The cell of claim 1, wherein the HLA-G or H2-M3 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:16 or SEQ ID NO:15. The cell of claim 1, wherein the Cd47 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4. The cell of claim 1, wherein the CD200 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6. The cell of claim 1, wherein the FASLG or FasL transgene encodes a protein that has at least 85% identity to the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO:9. The cell of claim 1, wherein the Ccl21 or Ccl21b transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:1. The cell of claim 1, wherein the Mfge8 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:13 or SEQ ID NO:14. The cell of claim 1, wherein the Serpin B9 or Spi6 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:7. The cell of claim 4, wherein the IFNγR1 d39 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:17. The cell of claim 1, wherein the two or more transgenes are operably linked to a constitutive promoter. The constitutive promoter is a CAG promoter, cytomegalovirus (CMV) promoter, EF1α promoter, PGK promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, HSV tk promoter, mouse mammary tumor virus (MMTV). 122. The cell of claim 121, which is selected from the group consisting of a promoter, an HIV LTR promoter, a Moloney virus promoter, an Epstein Barr Virus (EBV) promoter and a Rous sarcoma virus (RSV) promoter. The cell of claim 1, further comprising a transgene encoding a therapeutic agent. 124. The cell of claim 123, wherein the therapeutic agent is a protein or antibody. 125. The cell of claim 124, wherein the antibody is an inhibitory antibody or an agonist antibody. 124. The therapeutic agent of claim 123, wherein the therapeutic agent is the agent listed in Table 2 or a wild-type version of a gene known to be mutated in cancer, enzyme or hormone deficiency, metabolic disease or degenerative disease. cell. The therapeutic agent is an inducible expression selected from the group consisting of a tetracycline response element, a light induction system, a radioactive gene system, a cumate switch induction system, an ecdysone induction system, a destabilization domain system or a ligand reversible dimerization system. 124. The cell of claim 123, which is expressed using the system. The therapeutic agent is a CAG promoter, cytomegalovirus (CMV) promoter, EF1α promoter, PGK promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter. 124. The method of claim 123, which is expressed using a constitutive promoter selected from the group consisting of the HIV LTR promoter, the Moloney virus promoter, the Epstein-Barr virus (EBV) promoter and the Rous sarcoma virus (RSV) promoter. cell. A population of genetically modified cells comprising the cells of claim 1. A method for providing local immunosuppression at a transplant site in a homologous host, comprising transplanting the cell of claim 1 or the population of genetically modified cells of claim 129 to a transplant site in a homologous host. How to include. Following the transgene: TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, Dad1 and IFNganmaaru1 d39 or TGF-β, Cd73, Cd39, Lag3, Il1r2, Ackr2, Tnfrsf22, Tnfrs23, Tnfrsf10, 38. The method of claim 37, further comprising expressing one or more of the genes encoding biological agents that act as agonists of Dad1 or IFNγR1 d39. 132. The method of claim 131, wherein the TGF-β or biological agent acts locally within the implant environment. 38. The method of claim 37, wherein the cells are a source of stem cells, cells that are subject to genome editing and/or therapeutic cell types. The cells are embryonic stem cells, induced pluripotent stem cells, induced pluripotent stem cells (iPSC), hematopoietic stem cells, mesenchymal stem cells, endothelial stem cells, epithelial stem cells, adipose stem cells or progenitor cells, reproductive stem cells, lung stem cells or progenitor cells, Mammary gland stem cells, olfactory adult stem cells, hair follicle stem cells, intestinal stem cells or progenitor cells, pluripotent stem cells, amniotic stem cells, cord blood stem cells, neural stem cells or progenitor cells, adult stem cells, somatic stem cells, tissue-specific stem cells, totipotent stem cells , Fibroblast, monocyte progenitor cell, B cell, exocrine cell, pancreatic progenitor cell, endocrine progenitor cell, hepatoblast, myoblast, preadipocyte, hepatocyte, chondrocyte, smooth muscle cell, K562 human erythroid system Leukemia cell line, bone cell, synovial cell, tendon cell, ligament cell, meniscus cell, adipocyte, dendritic cell, natural killer cell, skeletal muscle cell, cardiomyocyte, erythroid-megakaryocytic cell, eosinophil , Macrophages, T cells, islet β cells, neurons, cardiomyocytes, blood cells, exocrine precursor cells, duct cells, acinar cells, α cells, β cells, δ cells, PP cells, cholangiocytes, white or brown adipocytes, Hormone-secreting cells, epidermal keratinocytes, epithelial cells, renal cells, germ cells, skeletal joint synovial cells, periosteal cells, perichondrocytes, chondrocytes, endothelial cells, pericardial cells, meningeal cells, keratinocyte precursor cells, keratinocytes Stem cells, pericytes, glial cells, ependymal cells, cells isolated from amnion or placental membrane, serosa cells, somatic cells or skin, heart, brain or spinal cord, liver, lung, kidney, pancreas, bladder, bone marrow, spleen 138. The method of claim 133, which is a cell derived from the intestine or the stomach. A method for controlling the growth of cells according to claim 1 at a transplant site in a homologous host, the method comprising:
a) genetically modifying a cell division locus (CDL) in a cell, said CDL being one or more endogenous or exogenous loci, the transcripts of which are expressed by dividing cells. One or more endogenous or exogenous loci, wherein the genetic modification of the CDL is
i) an ablation link (ALINK) system comprising a DNA sequence encoding a negative selectable marker transcriptionally linked to the DNA sequence encoding the CDL, and ii) an inducible activator operably linked to the CDL Genetic modification comprising one or more of the inducible exogenous activator of regulation of CDL (EARC) system, including a gene expression system based on
b) transplanting said cell or population of said cells at a transplant site in a homologous host, and c) maintaining said genetically modified cells comprising said ALINK system in the absence of an inducer of said negative selection marker. By allowing the growth of said genetically modified cell comprising said ALINK system and/or exposing said cell comprising said ALINK system to said inducer of said negative selection marker, said genetic modification comprising said ALINK system. Ablating and/or inhibiting cell proliferation, and/or ii) exposing the genetically modified cells comprising the EARC system to an inducer of a gene expression system based on the inducible activator. By allowing the growth of the genetically modified cells containing the EARC or maintaining the cells containing the EARC system in the absence of the inducer of the inducible activator-based gene expression system. A method comprising preventing or inhibiting the growth of said genetically modified cells comprising the system. 38. The method of claim 37, wherein the homologous host is a mammal. 138. The method of claim 136, wherein the allogeneic host is a mouse. 138. The method of claim 136, wherein the allogeneic host is a human. 38. The method of claim 37, wherein the host has a degenerative disease or condition that can be treated with cell therapy. The disease or condition is blindness, arthritis, ischemia, diabetes, multiple sclerosis, spinal cord injury, stroke, cancer, lung disease, blood disease, Parkinson's disease, Alzheimer's disease, Huntington's disease, ALS, enzyme or hormone deficiency, 140. The method of claim 139, which is a metabolic disease, autoimmune disease, age-related macular degeneration, retinal dystrophy, infection, hemophilia, myocardial infarction, degenerative disease or age-related disease. One or more of PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8 and Serpin B9 or Spi6 is above the expression level of the corresponding endogenous gene in activated leukocytes. 38. The method of claim 37, which is expressed at the level of. All eight of PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8 and Serpin B9 or Spi6 are above the expression level of the corresponding endogenous gene in activated leukocytes. 144. The method of claim 141, which is expressed at a level. 38. The method of claim 37, wherein the PD-L1 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:11 or SEQ ID NO:12. 38. The method of claim 37, wherein the HLA-G or H2-M3 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:16 or SEQ ID NO:15. 38. The method of claim 37, wherein the Cd47 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:4. 38. The method of claim 37, wherein the CD200 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6. 38. The method of claim 37, wherein the FASLG or FasL transgene encodes a protein that has at least 85% identity to the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO:9. 38. The method of claim 37, wherein the Ccl21 or Ccl21b transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:1. 38. The method of claim 37, wherein the Mfge8 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:13 or SEQ ID NO:14. 38. The method of claim 37, wherein the Serpin B9 or Spi6 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:9. 38. The method of claim 37, wherein the IFNγR1 d39 transgene encodes a protein having at least 85% identity to the amino acid sequence of SEQ ID NO:17. 38. The method of claim 37, wherein the one or more transgenes are operably linked to a constitutive promoter. The constitutive promoter is a CAG promoter, cytomegalovirus (CMV) promoter, EF1α promoter, PGK promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, HSV tk promoter, mouse mammary tumor virus (MMTV). 153. The method of claim 152, wherein the method is selected from the group consisting of a promoter, an HIV LTR promoter, a Moloney virus promoter, an Epstein Barr Virus (EBV) promoter and a Rous sarcoma virus (RSV) promoter. 38. The method of claim 37, wherein the cell further comprises a transgene encoding a therapeutic agent. 155. The method of claim 154, wherein the therapeutic agent is a protein or antibody. 156. The method of claim 155, wherein the antibody is an inhibitory antibody or an agonist antibody. 155. The method of claim 154, wherein the therapeutic agent is a drug listed in Table 2 or a wild-type version of a gene that is mutated in the subject. The therapeutic agent is an inducible expression selected from the group consisting of a tetracycline response element, a light induction system, a radioactive gene system, a cumate switch induction system, an ecdysone induction system, a destabilization domain system or a ligand reversible dimerization system. 155. The method of claim 154, which is expressed using the system. The therapeutic agent is a CAG promoter, cytomegalovirus (CMV) promoter, EF1α promoter, PGK promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter. 155. The method of claim 154, which is expressed using a constitutive promoter selected from the group consisting of the HIV LTR promoter, the Moloney virus promoter, the Epstein-Barr virus (EBV) promoter, and the Rous sarcoma virus (RSV) promoter. Method. A composition comprising the cells of claim 1. 166. The composition of claim 160, further comprising a pharmaceutically acceptable excipient. A kit comprising the cells of claim 1 or the composition of claim 160. A method of performing treatment of a disease or condition in a subject in need thereof, comprising administering to the subject the cells of claim 1 or the composition of claim 160. The disease or condition is blindness, arthritis, ischemia, diabetes, multiple sclerosis, spinal cord injury, stroke, cancer, lung disease, blood disease, Parkinson's disease, Alzheimer's disease, Huntington's disease, ALS, enzyme or hormone deficiency, 163. A metabolic disease, autoimmune disease, age-related macular degeneration, retinal dystrophy, infection, hemophilia, myocardial infarction, degenerative disease, age-related disease, or a disease or condition listed in Table 2, 163. The method described. 166. The method of claim 163, wherein the cells are differentiated into a lineage restricted cell type prior to administration to the subject. 166. The method of claim 165, wherein the disease or condition is myocardial infarction and the cells are differentiated into cardiomyocytes. 166. The method of claim 165, wherein the disease or condition is blind and the cells are differentiated into photoreceptor cells. 166. The method of claim 165, wherein the disease or condition is spinal cord injury, Parkinson's disease, Huntington's disease or Alzheimer's disease, and the cells are differentiated into neurons. 166. The method of claim 165, wherein the disease or condition is multiple sclerosis and the cells are differentiated into glial cells. 166. The method of claim 163, wherein the cells are locally administered to a tissue or body site in need of the cells or the therapeutic agent. 165. The method of claim 163, wherein the cells are administered intravenously, subcutaneously, intramuscularly, transdermally, intradermally, parenterally, intraarterially, intravascularly or by perfusion. 179. The method of claim 171, wherein the cells are administered by subcutaneous injection to produce cloaked subcutaneous tissue. 166. The method of claim 163, wherein the cells are administered as a tissue. 174. The method of claim 173, wherein the tissue is administered with a gel, biocompatible matrix or cell scaffold. 166. The method of claim 163, wherein the cells are administered in an amount of 25,000 to 5,000,000 cells. 179. The method of claim 171, wherein the cells are administered in an amount of 800,000,000 to 3,000,000 cells. 166. The method of claim 163, further comprising administering an additional therapeutic agent. 179. The method of claim 177, wherein the additional therapeutic agent is administered prior to administration of the cells. 179. The method of claim 177, wherein the additional therapeutic agent is administered after administration of the cells. 179. The method of claim 177, wherein the additional therapeutic agent is administered concurrently with the administration of the cells. The additional therapeutic agents include immunosuppressants, disease-modifying anti-rheumatic drugs (DMARDs), biological response modifiers (a type of DMARDs), corticosteroids or nonsteroidal anti-inflammatory drugs (NSAIDs), prednisone, prednisolone, Methylprednisolone, methotrexate, hydroxychloroquine, sulfasalazine, leflunomide, cyclophosphamide, azathioprine, tofacitinib, adalimumab, abatacept, anakinra, kinelet, certolizumab, etanercept, golimumab, infliximab, rituximab, 6-turizimab, tocilitumab, tocilicizumab, toricitumab, tocilicizumab, toricusimab, tocilitumab, tocilicizumab, toricusimab, or tociritumab, tocilicizumab, toricatimab, tocilicizumab, rituximab, or tociritumab Abatacept, adalimumab, alemtuzumab, aminosalicylate, antibiotics, antihistamine, anti-TNFα, azathioprine, belimumab, β-interferon, calcineurin inhibitor, certolizumab, corticosteroids, cromolyn, cyclosporin A, cyclosporine, dimethyl fumarate, etanermodin, etanermocept, , Fumarate, glatiramer acetate, golimumab, hydroxyurea, IFNγ, IL-11, leflunomide, leukotriene receptor antagonist, long-acting β2 agonist, mitoxantrone, mycophenolate mofetil, natalizumab, ocrelizumab, pimecrolimus, Probiotics, retinoids, salicylic acid, short-acting β2 agonists, sulfasalazine, tacrolimus, teriflunomide, theophylline, tocilizumab, ustekinumab or vedolizumab, bevacucizumab, ranibizumab or aflibercept), photodynamic therapy, photocoagulation, carbidopa-levodave Dopamine agonist, MAO-B inhibitor, catechol-O-methyltransferase inhibitor, anticholinergic, amantadine, deep brain stimulant, anticoagulant, antiplatelet agent, angiotensin converting enzyme inhibitor, angiotensin II receptor blocker , Angiotensin receptor neprilysin inhibitor, β blocker, combined α and β blocker, calcium channel blocker, cholesterol lowering drug, nicotinic acid, cholesterol absorption inhibitor, digitalis preparation, diuretic, vasodilator, antiplatelet agent Combination therapy, cardiac treatment, antiviral compound, nucleoside analog reverse transcriptase inhibitor (NRTI), non-nucleoside reverse transcriptase inhibitor (NNRTI), protease inhibitor, antibacterial compound, antifungal compound , Antiparasitic compound, insulin, sulfonylurea, biguanide, meglitinide, thiazolidinedione, DPP-4 inhibitor, SGLT2 inhibitor, α-glucosidase inhibitor, bile acid sequestrant, aspirin, diet, coagulation factor, desmopressin, blood clot Preservative, fibrin sealant, physical therapy, coenzyme, bone marrow transplant, organ transplant, hemodialysis, hemofiltration, exchange transfusion, peritoneal dialysis, medium chain triacylglycerol, miglustat, enzyme replacement therapy, checkpoint inhibitor, chemotherapy 179. The method of claim 177, which is a drug, biological agent, radiation therapy, cryotherapy, hyperthermia, surgical resection or tumor tissue or anti-cancer vaccine. 165. The method of claim 163, further comprising controlling proliferation of the cells. The cell comprises an ALINK system, and the method of controlling proliferation comprises
i) maintaining the cells containing the ALINK system in the absence of an inducer of the negative selectable marker to allow growth of the cells containing the ALINK system, or ii) containing the ALINK system 183. The method of claim 182, comprising ablating or inhibiting proliferation of the cells, including the ALINK system, by exposing the cells to the inducer of the negative selectable marker. The cell comprises an EARC system, and the method of controlling proliferation comprises
i) exposing the cells containing the EARC system to an inducer of a gene expression system based on the inducible activator to allow growth of the cells containing the EARC system, or ii) the EARC Maintaining or preventing the growth of said cells comprising said EARC system by maintaining said cells comprising said system in the absence of said inducer of said inducible activator-based gene expression system, Item 182. The method according to Item 182. 166. The method of claim 163, wherein the cells are removed after completion of the treatment. 160. The cell of claim 1 or the composition of claim 160, for use in treating a disease or condition in a subject in need thereof. The disease or condition is blindness, arthritis, ischemia, diabetes, multiple sclerosis, spinal cord injury, stroke, cancer, lung disease, blood disease, Parkinson's disease, Alzheimer's disease, Huntington's disease, ALS, enzyme or hormone deficiency, 187. A metabolic disease, autoimmune disease, age-related macular degeneration, retinal dystrophy, infection, hemophilia, myocardial infarction, degenerative disease, age-related disease, or a disease or condition listed in Table 2; The described cell. 160. The cell of claim 1 or the composition of claim 160, for use in providing local immunosuppression at a transplant site in a cognate host. One or more of PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8 and Serpin B9 or Spi6 are of gene expression for all genes in the cell's genome. 35. The cell of any one of claims 1-34, which is expressed at a level in the top 5%. All eight of PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8 and Serpin B9 or Spi6 are top of the gene expression for all genes in the genome of said cells. 35. The cell of any one of claims 1-34, which is expressed at a level at 5%. One or more of PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8 and Serpin B9 or Spi6 are of gene expression for all genes in the cell's genome. The cell according to any one of claims 1 to 3, which is expressed at a level in the top 5%. All eight of PD-L1, HLA-G or H2-M3, Cd47, Cd200, FASLG or FasL, Ccl21 or Ccl21b, Mfge8 and Serpin B9 or Spi6 are top of the gene expression for all genes in the genome of said cells. The cell according to any one of claims 1 to 3, which is expressed at a level at 5%.